Immunomodulating transgenic plants and related methods

ABSTRACT

The transgenic plants expressing one or more antagonist IL-10R peptides and anti-IL-10 single domain antibodies that stimulate or modulate the immune system and improve gastrointestinal physiology of an animal fed the transgenic plants or tissues thereof and the genes encoding the antagonist IL-10R peptides and anti-IL-10 single domain antibodies are described. The animal feed additives and animal feed incorporating the transgenic plants or tissues thereof are also described. Methods of stimulating or modulating an animal&#39;s immune system, improving an animal&#39;s gastrointestinal physiology, improving animal performance by using the transgenic plants or tissues thereof, and treating animals infected with a gastrointestinal pathogen are provided.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.16/609,633 filed on Oct. 30, 2019 as US National Stage of InternationalPatent Application No. PCT/US2018/034856, which was filed on May 29,2018, and claimed the benefit of U.S. provisional application No.62/512,444 filed May 30, 2017; all of which are incorporated herein byreference as if fully set forth.

The sequence listing electronically filed with this application titled“Sequence Listing,” which was created on May 19, 2022 and had a size of485,841 bytes is incorporated by reference herein as if fully set forth.

FIELD OF INVENTION

This disclosure relates to antagonist IL-10 receptor (IL-10R) peptidesand anti-IL-10 antibodies, including anti-IL-10 single domainantibodies. This disclosure relates to transgenic plants that expressand accumulate antagonist IL-10R peptides and anti-IL-10 single domainantibodies that stimulate or modulate the immune system and improvegastrointestinal physiology of an animal fed the transgenic plants ortissues thereof. This disclosure also relates to the genes encodingthese peptides and antibodies.

This disclosure relates to animal feed additives and animal feed thatincorporates the transgenic plants or tissues thereof including thepeptides and antibodies. This disclosure also relates to animal feedadditives and animal feed that incorporates the peptides and antibodies.

This disclosure relates to methods of treating animals infected with agastrointestinal pathogen by administering to them antagonist IL-10Rpeptides and anti-IL-10 single domain antibodies, transgenic plantsexpressing the peptides and antibodies disclosed herein, or feedinganimals with animal feed that includes these transgenic plants, peptidesor antibodies. The disclosure also relates to methods of stimulating ormodulating an animal's immune system, methods of improving an animal'sgastrointestinal physiology, methods of improving animal performance byusing the disclosed transgenic plants or tissues thereof, antagonistIL-10R peptides, or anti-IL-10 single domain antibodies.

This disclosure relates to methods of making antagonist IL-10R peptidesand anti-IL-10 single domain antibodies, and methods of makingtransgenic plants expressing the peptides and antibodies disclosedherein.

The sequence listing electronically filed with this application titled“Sequence Listing,” created on May 30, 2017, and having a file size of210,215 bytes is incorporated herein by reference as if fully set forth.

BACKGROUND

Coccidiosis is a common poultry disease caused by protozoan parasitesthat infect the gastrointestinal tract (Cervantes, H., 2002; Cervantes,H., 2006).

The disease spreads from one animal to another by contact with infectedfeces or ingestion of infected tissue. Coccidiosis in chickens is causedby infection of the intestinal lining cells by parasitic protozoa of thegenus Eimeria, and commonly by Eimeria tenella. The most commonmedications used to treat coccidial infections are anti-Coccidial drugs,antibiotics, and vaccines.

Anti-Coccidial drugs and Coccidiostats are used in poultry production tocontrol Coccidiosis and maintain animal productivity, which generallydecreases when animals are infected by Eimeria, and develop subclinicalor clinical Coccidiosis. Clinical Coccidiosis results in disruption ofthe digestive tract, and symptoms include weight loss, growthsuppression, diarrhea, bloody droppings and increased mortality.Subclinical Coccidiosis is common in poultry production, even whenemploying current Coccidiostats or vaccines, and does not present manyof the same symptoms as clinical Coccidiosis, but still decreases animalproductivity. The reduced animal productivity from Coccidiosis resultsin significant losses for the poultry industry, estimated at over onebillion US dollars per year.

Anti-Coccidial drugs, antibiotics, and vaccines are important forefficient poultry production, but are being phased out in many countriesdue to consumer concerns over their use and safety. Vaccine use ischallenged by incomplete immunity within the flock, and anti-Coccidialdrugs are costly, need to be administered at the right time and dose,and can lead to the development of resistant Eimeria strains. Industryhas witnessed a rise in the number of drug-resistant strains, whichlowers the value of these products and necessitates the development ofother methods for controlling Coccidiosis.

Eimeria stimulates production of an anti-inflammatory cytokineinterleukin 10 (IL-10). IL-10 interacts with its receptor IL-10R in theintestinal lining to suppress the immune response. In turn, this allowsEimeria infection to proceed without interference from the immunesystem. IL-10 is a potent anti-inflammatory cytokine that helps animalscontrol inflammation responses. IL-10 also controls the immune system toprevent hyper immune responses. Blocking IL-10 to prevent itsinteraction with IL-10R would prevent immune suppression, and thus,helps the animal's normal immune response to reduce and clear Eimeriainfection. In contrast to other prophylactic or therapeutic approachesto controlling Coccidiosis, blocking IL-10 suppression of the immunesystem should not lead to the development of resistant Eimeria strainsbecause such intervention focuses on stimulating the host's immuneresponse and not on attenuating or killing the infectious agent itself.

As previously described, this approach currently suffers fromsignificant limitations that have prevented widespread commercialadoption and industrial use. First, the antibodies used thus far havebeen generated by inoculating either a maternal hen, or eggs, with thetarget peptide. In the case of the former, only chicks from theinoculated hen may be used, requiring the inoculation of many hens forchick production, and full protection is not guaranteed due toinadequate immunity, an ineffective peptide (stimulating antigen), or anunprolonged response. Peptide effectiveness may also be challenged sinceit is well known that small peptides often do not mobilize an effectiveimmune response, and because IL-10 (or IL-10 homologs) is produced bythe host, it may be difficult to generate adequate antibodies withoutthe use of adjuvants or conjugates, which further increases the cost andcomplexity of this approach. Furthermore, because IL-10 is known todimerize in vivo, selected peptides may generate antibodies to epitopesthat are not normally exposed by the IL-10 dimer and therefore may beineffective in binding IL-10 in the host animal. Likewise, inoculatingeggs (or collecting eggs from inoculated hens) is cumbersome andincreases costs, suffers from many of the same issues that challenge heninoculation, and adds additional costs when the antibodies must beharvested from the yolks. In the case where the antibodies are harvestedfrom the eggs, the material must be dried, stabilized, and then mixedinto feed to deliver to chicks. While the added processing steps(including harvesting the eggs, drying, formulating and packaging forfeed addition), add extra cost, it is unclear how consistent thisproduction method will be, how susceptible it is to contamination byother infectious agents, or whether the antibodies generated in thismanner will be thermally stable enough to survive the pelletingprocesses used in preparing animal feed. Many full-length antibodies donot possess the thermal stability required to maintain their solubility,structure, and affinity for IL-10, when combined with animal feed andprocessed through a pellet mill. Antibodies delivered in pelleted feedwill be exposed to pelleting temperatures that may be 65° C., 70° C.,75° C., 80° C., 82° C., 85° C., 90° C., 95° C., or even greater. Forthese reasons, using eggs to produce antibodies for animal feed is avery challenging, high-cost practice, and because the antibodies arenever fully sequenced or characterized, this production method precludesthe use of biotechnology to improve antibody properties and the cost,efficiency, and efficacy of production. Therefore, new technologies aregreatly needed if modulation of the IL-10 signaling pathway is toachieve any market relevance in the animal production industry. Toaddress these shortcomings, there exists a need for a novel, low-costfeed additive that ideally is delivered in existing diet ingredients,that has increased thermal stability to endure the feed pelletingprocess, and that can more effectively inhibit the IL-10 signalingprocess.

SUMMARY

In an aspect, the invention relates to a transgenic plant or tissuesthereof comprising a synthetic polynucleotide encoding at least oneantagonist IL-10R peptide, or an anti-IL-10 single domain antibody.

In an aspect, the invention relates to at least one antagonist IL-10Rpeptide. The at least one antagonist IL-10R peptide is one peptidecomprising an amino acid sequence with at least 90% identity to areference sequence selected from the group consisting of: SEQ ID NOS:1-13. The at least one antagonist IL-10R peptide comprises concatenatedpeptides comprising an amino acid sequence with at least 90% identity toa reference sequence selected from the group consisting of: SEQ ID NOS:32-40.

In an aspect, the invention relates to a synthetic polynucleotideencoding the at least one IL-10R antagonist peptide described herein.

In an aspect, the invention relates to an anti-IL-10 single domainantibody that binds to a polypeptide comprising an amino acid sequenceof SEQ ID NO: 80.

In an aspect, the invention relates to a synthetic polynucleotideencoding any one of the anti-IL-10 single domain antibodies describedherein.

In an aspect, the invention relates to an animal feed comprising any oneof the transgenic plants or tissues thereof described herein.

In an aspect, the invention relates to an animal feed comprising any ofthe antagonist IL-10R peptides, or anti-IL-10 single domain antibodiesdescribed herein.

In an aspect, the invention relates to a method of treating orpreventing a gastrointestinal infection in an animal comprising feedingthe animal any one of the transgenic plants or tissues thereof,antagonist IL-10R peptides, anti-IL-10 single domain antibodies, oranimal feed described herein.

In an aspect, the invention relates to a method of stimulating ormodulating the immune system and improving gastrointestinal physiologyof an animal comprising feeding the animal any one of the transgenicplants or tissues thereof, antagonist IL-10R peptides, anti-IL-10 singledomain antibodies or the animal feed described herein.

In an aspect, the invention relates to a method of improving animalperformance comprising feeding an animal any one of the transgenicplants or tissues thereof, antagonist IL-10R peptides, anti-IL-10 singledomain antibodies or animal feed described herein.

In an aspect, the invention relates to a method of preparing an animalfeed comprising mixing any one of the transgenic plants or tissuesthereof described herein with plant material to form a mixture.

In an aspect, the invention relates to a method of preparing an animalfeed comprising mixing any one of the antagonist IL-10R peptides oranti-IL-10 single domain antibodies described herein with plant materialto form a mixture.

In an aspect, the invention relates to a method of preparing atransgenic plant or tissues thereof comprising any of the antagonistIL-10R peptides or anti-IL-10 single domain antibodies described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of thepresent invention will be better understood when read in conjunctionwith the appended drawings. For the purpose of illustrating theinvention, particular embodiments are shown in the drawings. It isunderstood, however, that the invention is not limited to the precisearrangements and instrumentalities shown. In the drawings:

FIGS. 1A-1G are schematic drawings of the vectors pAG4305 (FIG. 1A),pAG4306 (FIG. 1B), pAG4308 (FIG. 1C), pAG4310 (FIG. 1D), pAG4311 (FIG.1E), pAG4312 (FIG. 1F), and pAG4313 (FIG. 1G).

FIGS. 2A-2D are schematic drawings of the vectors pAG4981 (FIG. 2A),pAG4982 (FIG. 2B), pAG4983 (FIG. 2C), and pAG4984 (FIG. 2D).

FIG. 3 illustrates the antibody response generated by a llama injectedwith full-length chicken IL-10. It demonstrates that specific antibodiesare produced by the animal that increases the binding of chicken IL-10post-injection, relative to the pre-immune (that is, pre-injection)state of the animal.

FIG. 4 illustrates the sequencing results from the anti-IL-10 antibodydevelopment and inter-relationship among the identified sequences. Inthis figure, sequences of sdAbs according to embodiments herein arealigned and compared to the sequence of sdAbs 40-IL-bR2-1115 set forthas SEQ ID NO: 208. The amino acid (AA) positions replaced in thesequence of SEQ ID NO: 208 are as follows: for 68-IL-bR2-1D9, AA 26 to34 (SEQ ID NO: 209); for 80-IL-bR2-1H10, AA 54 to 57 (SEQ ID NO: 210);for 03-IL-bR2-1C1, 04-IL-bR2-1D1, 81-IL-bR2-1A11, 63-IL-bR2-1G8, and33-IL-bR2-1A5, AA 30 to 34 (SEQ ID NO: 211), AA 97 to 103 (SEQ ID NO:212), and AA 105 to 112) SEQ ID NO: 213); for 35-IL-bR2-1C5, AA 30 to 34(SEQ ID NO: 214), AA 50 to 54 (SEQ ID NO: 215), and AA 97 to 103 (SEQ IDNO: 212); for 48-IL-bR2-1E16, AA 27 to 34 (SEQ ID NO: 216), AA 75 to 79(SEQ ID NO: 217), and AA 105 to 112 (SEQ ID NO: 218); for 01-IL-bR2-2A8and 70-IL-bR2-1F9, AA 27 to 34 (SEQ ID NO: 216), AA 105 to 112 (SEQ IDNO: 218), and AA 76 to 79 (SEQ ID NO: 219); for 85-IL-bR2-1E11, AA 102to 108 (SEQ ID NO: 220); for 44-IL-bR2-1D6, AA 26 to 34 (SEQ ID NO:221), AA 99 to 102 (SEQ ID NO: 222), and AA 104 to 113 (SEQ ID NO: 223);for 27-IL-bR2-1C4, AA 27 to 31 (SEQ ID NO: 224), AA 46 to 50 (SEQ ID NO:225), AA 53 to 61 (SEQ ID NO: 226), and AA 98 to 105 (SEQ ID NO: 227);for 32-IL-bR2-1114, AA 52 to 57 (SEQ ID NO: 228), and AA 98 to 102 (SEQID NO: 229); for 86-IL-bR2-1F11, AA 100 to 104 (SEQ ID NO: 230), and AA107 to 110 (SEQ ID NO: 231); for 20-IL-bR2-ID3, AA 44 to 47 (SEQ ID NO:232), AA 52 to 55 (SEQ ID NO: 233), and AA 100 to 107 (SEQ ID NO: 234);for 49-IL-bR2-1A7, AA 27 to 35 (SEQ ID NO: 235), and AA 97 to 109 (SEQID NO: 236); for 24-IL-bR2-1113, AA 24 to 37 (SEQ ID NO: 237), AA 98 to101 (SEQ ID NO: 238), and AA 103 to 108 (SEQ ID NO: 239); for58-IL-bR2-1B8, AA 26 to 35 (SEQ ID NO: 240), AA 46 to 61 (SEQ ID NO:241), and AA 97 to 107 (SEQ ID NO: 242); for 10-IL-bR2-1B2, AA 27 to 32(SEQ ID NO: 243), AA 52 to 59 (SEQ ID NO: 244), AA 75 to 80 (SEQ ID NO:245), and AA 97 to 104 (SEQ ID NO: 246); for 12-IL-bR2-1D2, AA 30 to 34(SEQ ID NO: 247), and AA 55 to 58 (SEQ ID NO: 248); and for76-IL-bR2-1D10, AA 99 to 103 (SEQ ID NO: 249).

FIG. 5 illustrates apparent binding affinity of anti-IL-10 antibodies tochicken IL-10.

FIG. 6 illustrates results of the anti-IL-10 antibody digestion in thesimulated gastric fluid (SGF) test.

FIG. 7 illustrates the apparent inhibition of IL-10 binding to the IL-10receptor in the presence of anti-IL10 antibodies chIL10sdAB1A11 (SEQ IDNO: 135) and chIL10sdAB1F11 (SEQ ID NO: 146).

FIG. 8 illustrates IL-10 suppression of Concanavalin A-induced secretionof IFN-γ secretion in primary chicken spleen cells.

FIG. 9 illustrates recovery of IFN-γ secretion from primary chickenspleen cells treated with Concanavalin A and chicken IL-10, when alsotreated with the anti-IL-10 antibodies (chIL10sdAB1A11 (SEQ ID NO: 135),chIL10sdAB1B9 (SEQ ID NO: 111), and chIL10sdAB1F11 (SEQ ID NO: 146)).

FIG. 10A is a schematic drawing of the vector pAG4314.

FIG. 10B is a schematic drawing of the vector pAG4988.

FIG. 11 illustrates that single domain antibodies express at high levelin individual corn grain. In both transgenic events, individual grainwere genotyped and protein extracted. The presence of the sdAB band inthe gel image correlates perfectly with the presence of the sdAB gene asrepresented at the top of the figure with a “+” if the sdAB gene ispresent and a “−” if the sdAB gene is absent.

FIG. 12 is a schematic drawing of the pLH1A11int expression cassette.

FIG. 13 is a schematic drawing of the pLH1A11 expression cassette.

FIG. 14 illustrates end point RT-PCR analysis of transiently expressedNb1A11 in N. benthamiana leaves. Lanes 1-5 contain the followingsamples: lane 1—GV3101+pLH9000 (negative control); lane2—GV3101+pLH1A11int; lane 3—GV3101+pLH1A11; lane 4—plasmid pLH1A11int;and lane 5—plasmid pLH1A11.

FIG. 15 illustrates sdAB1A11 expression in Agrobacterium infiltratedleaves of N. benthamiana. The Western blot shows detection of sdAB1A11in samples 5 and 6.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Certain terminology is used in the following description for convenienceonly and is not limiting.

“Synthetic nucleic acid sequence,” “synthetic polynucleotide,”“synthetic oligonucleotide,” “synthetic DNA,” or “synthetic RNA” as usedherein refers to a nucleic acid sequence, a polynucleotide, anoligonucleotide, DNA, or RNA that differs from one found in nature byhaving a different sequence that one found in nature or a chemicalmodification not found in nature. The definition of synthetic nucleicacid includes but is not limited to a DNA sequence created usingbiotechnology tools. Such tools include but are not limited torecombinant DNA technology, chemical synthesis, or directed use ofnucleases (so called “genome editing” or “gene optimizing”technologies).

“Synthetic protein,” “synthetic polypeptide,” “synthetic oligopeptide,”or “synthetic peptide” as used herein refers to a protein, polypeptide,oligopeptide or peptide that was made through a synthetic process. Thesynthetic process includes but is not limited to chemical synthesis orrecombinant technology.

As used herein, the terms “interleukin 10,” “IL10” and “IL-10” are usedinterchangeably, and refer to cytokine synthesis inhibitory factor,i.e., an anti-inflammatory cytokine. The terms “cIL-10,” “cIL10”,“chIL10”, and “chIL-10” refer to the chicken interleukin 10.

As used herein, the terms “antagonist IL-10R peptide,” “antagonistIL10R,” “IL10R antagonist peptide,” and “IL-10R antagonist peptide” areused interchangeably, and refer to peptides that are inhibitors of IL-10receptors (IL-10R). The IL-10R antagonist peptides may be fragments ofIL-10, or may differ from the fragments of IL-10. The IL-10R antagonistpeptide may be an antagonist derived from the IL-10R. The IL-10Rantagonist peptide may be fusion of the peptides, concatenation of thepeptides, or any other peptides that are capable of blocking orantagonizing IL-10 receptors. The IL-10R antagonist peptides can blockor antagonize receptors in any way, e.g., by blocking the IL-10 bindingpockets of the IL-10 receptors, preventing IL-10 from binding to thereceptors, blocking IL-10 dimerization, or IL-10 receptor assembly, orallowing IL-10 binding to the receptors but blocking subsequent signaltransduction. The IL-10R antagonist peptides can block or antagonizeIL-10 receptors by any mechanism or mode of action.

“Antibody” as used herein refers to an immunoglobulin molecule whichspecifically binds with an antigen.

“Synthetic antibody” as used herein refers to an antibody which isgenerated using recombinant DNA technology, such as, for example, anantibody expressed by a host engineered to produce the antibody, such asa mammalian cell, microbial cell, or plant as described herein. The termshould also be construed to mean an antibody which has been generated bythe synthesis of a DNA molecule encoding the antibody and which DNAmolecule expresses an antibody protein, or an amino acid sequencespecifying the antibody, wherein the DNA or amino acid sequence has beenobtained using synthetic DNA or amino acid sequence technology which isavailable and well known in the art. A “synthetic antibody” describedherein may include fragments and hybrids of antibodies. A “syntheticantibody” described herein may be generated by an organism that is dosedwith a specific antigen, and the antibody generated by the organism isisolated and propagated in a second organism.

A “single domain antibody,” or sdAB, refers to a synthetic antibody thatis a small monomeric antigen-binding fragment of an antibody, i.e., thevariable region of an antibody heavy or light chain. sdABs can bederived from antibodies that occur naturally or are generated incamelids, e.g., camels, and llamas, and may be produced by immunizing acamelid with a target antigen, isolating peripheral bloodmononucleocytes, isolating their nucleic acids, and cloning sdAB codingregions from specific nucleic acid fragments. sdABs may be also producedin cell culture, by microbial hosts in a fermentation process, or byplants. An antibody described herein may be a sdAB comprising a VHHdomain substantially as set out herein. A single domain antibody is asynthetic antibody.

“Antigen” as used herein is defined as a molecule that triggers animmune response. The immune response may involve either antibodyproduction, or the activation of specific immunologically active cells,or both. The antigen may refer to any molecule capable of stimulating animmune response, including macromolecules such as proteins or peptides.The antigen may be synthesized, produced recombinantly in a mammalian,insect, microbial or plant cell, or may be derived from a biologicalsample, including but not limited to a tissue sample, a cell, or abiological fluid.

“Binding affinity” refers to the sum total noncovalent interactionbetween members of binding pairs, e.g., an antibody and antigen. Thebinding affinity of the antibody can be determined based on apparentbinding EC50 value. As used herein, the term “EC50” or “EC₅₀” refers tothe half maximal effective concentration, which includes theconcentration of an antibody which induces a response halfway betweenthe baseline and maximum after a specified exposure time. The EC₅₀essentially represents the concentration of an antibody where 50% of itsmaximal effect is observed. In certain embodiments, the EC₅₀ valueequals the concentration of an antibody of the invention that giveshalf-maximal binding to cells expressing chicken IL-10, as determinedby, e.g., an ELISA assay. Thus, reduced or weaker binding is observedwith an increased EC₅₀, or half maximal effective concentration value.

As used herein, “variant” refers to a protein or DNA molecule that hasan amino acid or nucleic acid sequence that differs from the originalsequence but retains a biological activity that is the same orsubstantially similar to that of the original sequence. The variant maybe from the same or different species or be a synthetic sequence basedon a natural or prior molecule.

The words “a” and “one,” as used in the claims and in the correspondingportions of the specification, are defined as including one or more ofthe referenced item unless specifically stated otherwise. Thisterminology includes the words above specifically mentioned, derivativesthereof, and words of similar import. The phrase “at least one” followedby a list of two or more items, such as “A, B, or C,” means anyindividual one of A, B or C as well as any combination thereof.

In an embodiment, one or more antagonist IL-10R peptides is provided.The antagonist IL-10R peptide may be expressed separately as oneantagonist IL-10R peptide. The antagonist IL-10R peptide may include anamino acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93,94, 95, 96, 97, 98, 99 or 100% identity to a reference sequence selectedfrom the group consisting of: SEQ ID NO: 1 [P21], SEQ ID NO: 2 [P22],SEQ ID NO: 3 [P23], SEQ ID NO: 4 [P24], SEQ ID NO: 5 [P25], SEQ ID NO: 6[P26], SEQ ID NO: 7 [P27], SEQ ID NO: 8 [P28], SEQ ID NO: 9 [P29], SEQID NO: 10 [P11], SEQ ID NO: 11 [P30], SEQ ID NO: 12 [P31], and SEQ IDNO: 13 [P32].

An antagonist IL-10R peptide may be expressed in the form ofconcatenated antagonist IL-10R peptides. The concatenated antagonistIL-10R peptides may comprise a first antagonist IL-10R peptide having anamino acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93,94, 95, 96, 97, 98, 99 or 100% identity to a reference sequence selectedfrom the group consisting of SEQ ID NOS: 1-13 fused to a secondantagonist IL-10R peptide having an amino acid sequence with at least70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identity to a reference sequence selected from the group consisting ofSEQ ID NOS: 1-13. The first antagonist IL-10R peptide may differ fromthe second antagonist IL-10R peptide. The first antagonist IL-10Rpeptide may be similar to the second antagonist IL-10R peptide. Theconcatenated antagonist IL-10R peptides may have more than twoantagonist IL-10R peptides. Each of the first antagonist IL-10R peptideand the second antagonist IL-10R peptide included in the concatenatedantagonist IL-10R peptides may have an amino acid sequence with at least70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identity to a reference sequence selected from the group consisting ofSEQ ID NOS: 1-13. Subsequent antagonist IL-10R peptides may differ fromthe first and second antagonist IL-10R peptides and from each other.Subsequent antagonist IL-10R peptides may be similar to the first andthe second antagonist IL-10R peptide and to each other. The concatenatedantagonist IL-10R peptides may comprise an amino acid sequence with atleast 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identity to a reference sequence selected from the group consisting ofSEQ ID NO: 32 [P2501], SEQ ID NO: 33 [P2502], SEQ ID NO: 34 [P2503], SEQID NO: 35 [P2504], SEQ ID NO: 36 [P2505], SEQ ID NO: 37 [P2506], SEQ IDNO: 38 [P2507], SEQ ID NO: 39 [P2508], and SEQ ID NO: 40 [P2509]. Thefirst antagonist IL-10R peptide may be linked to the second antagonistIL-10R peptide by a linker. Each of the first, the second and thesubsequent antagonist IL-10R peptides may be linked to each other by oneor more linkers. The one or more linker may be selected from the groupconsisting of SEQ ID NOS: 41-44, and 65. The antagonist IL-10R peptideor the concatenated antagonist IL-10R peptides may comprise a signalpeptide. The signal peptide may be but is not limited to an amyloplasttargeting signal, a cell wall targeting peptide, a mitochondrialtargeting peptide, a cytosol localization signal, a chloroplasttargeting signal, a nuclear targeting peptide, or a vacuole targetingpeptide. The signal peptide may an N-terminal signal peptide or aC-terminal signal peptide. The N-terminal signal peptide may be but isnot limited to OsGluB4sp (rice Glu-B4 glutelin signal peptide), BAASS(barley alpha amylase signal sequence), PR1 (pathogenesis relatedprotein), or zein 27 (xGZm27ss) signal peptide. The C-terminal signalpeptide may be but is not limited to KDEL (SEQ ID NO: 29), HDEL (SEQ IDNO: 30), SEKDEL (SEQ ID NO: 31), HvVSD from barley polyamine oxidase, orHvAle from barley aleurone (thiol protease). The IL-10R antagonistpeptide or the concatenated IL-10R antagonist peptides may be fused tothe N-terminal signal peptide or C-terminal signal peptide, or both.

The antagonist IL-10R peptide, or the concatenated antagonist IL-10Rpeptides may be capable of reducing IL-10 binding to the IL-10R. Theantagonist IL-10R peptide, or the concatenated antagonist IL-10Rpeptides may decrease the production of interferon gamma or nitric oxidewhen used in a cellular assay comprising cells that are stimulated byIL-10 to increase production of interferon gamma or nitric oxide.

In an embodiment, the antagonist IL-10R peptide having less than 100%identity to its corresponding amino acid sequence of SEQ ID NO: 1-13 or32-40 may be a variant of the referenced peptide or amino acid. In anembodiment, an isolated peptide having a sequence with at least 70, 75,80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to apeptide having the sequence of any one of SEQ ID NOS: 1-13 and 32-40along 7 to 10, 7 to 15, 7 to 30, 7 to 40, 7 to 50, or 7 to all aminoacids of a protein having the sequence of any of one any one of SEQ IDNOS: 1-13 and 32-40 is provided. This list of sequence lengthsencompasses every full length peptide in SEQ ID NOS: 1-13 and 32-40 andevery smaller length within the list, even for peptides that do notinclude over 50 amino acids. For example, the lengths of 7 to 10, 7 to20, 7 to 30, and 7 to all amino acids would apply to a sequence with 50amino acids. A range of amino acid sequence lengths recited hereinincludes every length of amino sequence within the range, endpointsinclusive. The recited length of amino acids may start at any singleposition within a reference sequence where enough amino acids follow thesingle position to accommodate the recited length. The fragment of theantagonist IL-10R peptide may be a subsequence of the polypeptidesherein that retain at least 40% of the antagonist IL-10R peptide EC₅₀value when used in a cellular assay comprising cells that are inhibitedby IL-10 (in the presence of ConA or PHA) to decrease production ofinterferon gamma or nitric oxide. The fragment may have 5, 7, 8, 9, 10,15, 20, 25, 30, 35, 40, 45 or 50 amino acids. The fragments may include5, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 contiguous amino acids.Embodiments also include nucleic acids or polynucleotides, encoding saidamino acid sequences. A less than full length amino acid sequence may beselected from any portion of one of the sequences of SEQ ID NOS: 1-13and 32-40 corresponding to the recited length of amino acids. A lessthan full length amino acid sequence may be selected from a portion ofany one of SEQ ID NOS: 1-13 and 32-40.

In an embodiment, an antagonist IL-10R peptide or concatenatedantagonist IL-10R peptides may maintain or improve the body weight, and,or the feed conversion ratio, of poultry fed the peptides relative topoultry fed the same feed lacking the peptides. The antagonist IL-10Rpeptide or concatenated antagonist IL-10R peptides may be dosed at lessthan 500 mg per kg of pelleted feed, or more preferably at less than 50mg per kg of pelleted feed, or even more preferably at less than 5 mgper kg of pelleted feed, or even more preferably at less than 1 mg perkg of pelleted feed. The antagonist IL-10R peptide or concatenatedantagonist IL-10R peptides may also improve the body weight or feedconversion ratio of poultry when used in mash (non-pelleted) feed. Theantagonist IL-10R peptide or concatenated antagonist IL-10R peptides maymaintain their affinity for the IL-10R following incubation in liquidfor at least 60 seconds at a temperature less than or equal to 65° C.,or 70° C., or 75° C., or 80° C., or 85° C., or 90° C., or 95° C., or100° C. The antagonist IL-10R peptide or concatenated antagonist IL-10Rpeptides may maintain their affinity for the IL-10R when heated to atemperature of 70° C. to 90° C. The antagonist IL-10R peptide orconcatenated antagonist IL-10R peptides may maintain their affinity forthe IL-10R when heated to a temperature in a range between any two ofthe following values: 70° C., 75° C., 80° C., 85° C., or 90° C.

In an embodiment, the antagonist IL-10R peptide or concatenatedantagonist IL-10R peptides may be a peptide or concatenated peptidesthat are stable to pepsin digestion, may have an increased stability inthe animal digestive tract, and may be produced by a microbial host. Theantagonist IL-10R peptide or concatenated antagonist IL-10R peptides maybe a peptide or concatenated peptides that are readily degradable bypepsin. The readily degradable peptide or concatenated peptides maycompletely degrade in a time period from 45 minutes to 40 minutes, from40 minutes to 35 minutes, from 35 minutes to 30 minutes, from 30 minutesto 25 minutes, from 25 minutes to 20 minutes, from 20 minutes to 15minutes, from 15 minutes to 10 minutes, from 10 minutes to 8 minutes,from 8 minutes to 6 minutes, from 6 minutes to 4 minutes, from 4 minutesto 2 minutes of the pepsin treatment. The time period for degradationmay be in a range between any two integer value between 2 minutes and 45minutes. The complete degradation of the peptide or concatenatedpeptides by pepsin may occur in 10 minutes.

An embodiment provides an antibody that binds to IL-10, and is referredherein as anti-IL-10 antibody. The anti-IL-10 antibody may bind to theGallus gallus (chicken) IL-10, and is referred herein as an anti-chIL-10antibody, or chIl10AB. The anti-IL-10 antibody may be a single domainanti-IL-10 antibody (sdAB). Both “single domain anti-IL-10 antibody” and“anti-IL-10 single domain antibody” refer to the same type of antibodyand may be used interchangeably herein. An anti-IL-10 single domainantibody may bind to a polypeptide comprising an amino acid sequence ofSEQ ID NO: 80. The anti-IL-10 single domain antibody may be capable ofreducing IL-10 binding to the IL-10 receptor (IL-10R).

In an embodiment, the anti-IL-10 single domain antibody may have abinding EC₅₀ for chicken I1-10 of 30 nM, or less. The anti-IL-10 singledomain antibody may have a binding EC50 for chicken IL-10 of about 30nM, or less, about 25 nM, or less, about 20 nM, or less, about 15 nM, orless, about 10 nM, or less, about 5 nM, or less, or about 1 nM, or less.The anti-IL-10 single domain antibody may have a binding EC₅₀ forchicken I1-10 in a range between any two of the following EC₅₀ values:30, 20, 10, 5, or 1 nM. In an embodiment, the EC₅₀ of the anti-IL-10single domain antibody provided herein may be measured by ELISA or anyother assay known in the art. The EC₅₀ of the anti-IL-10 single domainantibody value may be measured by an ELISA assay described in Example 9herein.

Without limitations, the anti-IL-10 single domain antibody may be asingle domain antibody of any length and of any molecular mass that iscapable of reducing IL-10 binding to the IL-10 receptor (IL-10R). Theanti-IL-10 single domain antibody may have a molecular mass of 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 kDa. The anti-IL-10 single domainantibody may have a molecular mass of 14, 15, 16, 17, 18, 19, or 20 kDa.The anti-IL-10 single domain antibody may have a molecular mass in arange between any two of the following molecular masses: 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 kDa. The anti-Il-10 single domain antibodymay have a molecular mass in a range between any two of the followingmolecular masses: 14, 15, 16, 17, 18, 19, or 20 kDa.

The anti-IL-10 single domain antibody may include an amino acid sequencewith at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100% identity to a reference sequence selected from the groupconsisting of: SEQ ID NO: 87 [chIL10sdAB1H5], SEQ ID NO: 88[chIL10sdAB1E9], SEQ ID NO: 89 [chIL10sdAB1H1], SEQ ID NO: 90[chIL10sdAB1G6], SEQ ID NO: 91 [chIL10sdAB1C10], SEQ ID NO: 92[chIL10sdAB1B6], SEQ ID NO: 93 [chIL10sdAB1D12], SEQ ID NO:947[chIL10sdAB1C2], SEQ ID NO: 95 [chIL10sdAB1B5], SEQ ID NO: 96[chIL10sdAB1E2], SEQ ID NO: 97 [chIL10sdAB1G7], SEQ ID NO: 98[chIL10sdAB1G9], SEQ ID NO: 99 [chIL10sdAB1H12], SEQ ID NO: 100[chIL10sdAB2A9], SEQ ID NO: 101 [chIL10sdAB1E12], SEQ ID NO: 102[chIL10sdAB1E10], SEQ ID NO: 103 [chIL10sdAB1F12], SEQ ID NO: 104[chIL10sdAB1A8], SEQ ID NO: 105 [chIL10sdAB1C8], SEQ ID NO: 106[chIL10sdAB1C12], SEQ ID NO: 107 [chIL10sdAB1B1], SEQ ID NO: 108[chIL10sdAB1F1], SEQ ID NO: 109 [chIL10sdAB1D11], SEQ ID NO: 110[chIL10sdAB1E6], SEQ ID NO: 111 [chIL10sdAB1B9], SEQ ID NO: 112[chIL10sdAB1B10], SEQ ID NO: 113 [chIL10sdAB1F5], SEQ ID NO: 114[chIL10sdAB1A6], SEQ ID NO: 115 [chIL10sdAB1D5], SEQ ID NO: 116[chIL10sdAB1D8], SEQ ID NO: 117 [chIL10sdAB1B4], SEQ ID NO: 118[chIL10sdAB1C7], SEQ ID NO: 119 [chIL10sdAB1B3], SEQ ID NO: 120[chIL10sdAB1D7], SEQ ID NO: 121 [chIL10sdAB1F7], SEQ ID NO: 122[chIL10sdAB1F10], SEQ ID NO: 123 [chIL10sdAB1F2], SEQ ID NO: 124[chIL10sdAB1F3], SEQ ID NO: 125 [chIL10sdAB1F8], SEQ ID NO: 126[chIL10sdAB1C9], SEQ ID NO: 127 [chIL10sdAB1A12], SEQ ID NO: 128[chIL10sdAB1C3], SEQ ID NO: 129 [chIL10sdAB1E7], SEQ ID NO: 130[chIL10sdAB1D9], SEQ ID NO: 131 [chIL10sdAB1A9], SEQ ID NO: 132[chIL10sdAB1H10], SEQ ID NO: 133 [chIL10sdAB1C1], SEQ ID NO: 134[chIL10sdAB1D1], SEQ ID NO: 135 [chIL10sdAB1A11], SEQ ID NO: 136[chIL10sdAB1G8], SEQ ID NO: 137 [chIL10sdAB1A5], SEQ ID NO: 138[chIL10sdAB1C5], SEQ ID NO: 139 [chIL10sdAB1H6], SEQ ID NO: 140[chIL10sdAB2A8], SEQ ID NO: 141 [chIL10sdAB1F9], SEQ ID NO: 142[chIL10sdAB1E11], SEQ ID NO: 143 [chIL10sdAB1D6], SEQ ID NO: 144[chIL10sdAB1C4], SEQ ID NO: 145 [chIL10sdAB1H4], SEQ ID NO: 146[chIL10sdAB1F11], SEQ ID NO: 147 [chIL10sdAB1D3], SEQ ID NO: 148[chIL10sdAB1A7], SEQ ID NO: 149 [chIL10sdAB1H8], SEQ ID NO: 150[chIL10sdAB1H3], SEQ ID NO: 151 [chIL10sdAB1B8], SEQ ID NO: 152[chIL10sdAB1B2], SEQ ID NO: 153 [chIL10sdAB1D2], and SEQ ID NO: 154[chIL10sdAB1D10]. The anti-IL-10 single domain antibody may be fused toa signal peptide. The signal peptide may be but is not limited to anamyloplast targeting signal, a cell wall targeting peptide, amitochondrial targeting peptide, a cytosol localization signal, achloroplast targeting signal, a nuclear targeting peptide, anendoplasmic reticulum retention signal, or a vacuole targeting peptide.The signal peptide may an N-terminal signal peptide or a C-terminalsignal peptide. The N-terminal signal peptide may be but is not limitedto OsGluB4sp (rice Glu-B4 glutelin signal peptide), BAASS (barley alphaamylase signal sequence), PR1 (pathogenesis related protein), or zein 27(xGZm27ss) signal peptide. The C-terminal signal peptide may be but isnot limited to KDEL (SEQ ID NO: 29), HDEL (SEQ ID NO: 30), SEKDEL (SEQID NO: 31), HvVSD from barley polyamine oxidase, or HvAle from barleyaleurone (thiol protease). The anti-IL-10 single domain antibody may befused to the N-terminal signal peptide or C-terminal signal peptide, orboth.

In an embodiment, the anti-IL-10 single domain antibody having less than100% identity to its corresponding amino acid sequence of one of SEQ IDNO: 87-154 may be a variant of the referenced peptide or amino acid. Inan embodiment, an isolated peptide having a sequence with at least 70,75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity toa peptide having the sequence of any one of SEQ ID NOS: 87-154 along 10to 20, 10 to 25, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to80, 10 to 90, 10 to 100, 10 to 110, 10 to 115, 10 to 116, 10 to 117, 10to 118, 10 to 119, 10 to 120, 10 to 121, 10 to 122, 10 to 123, 10 to124, 10 to 125, 10 to 126, or 10 to all amino acids of a protein havingthe sequence of any of one any one of SEQ ID NOS: 87-154 is provided.This list of sequence lengths encompasses every full length peptide inSEQ ID NOS: 87-154 and every smaller length within the list, even forpeptides that do not include over 126 amino acids. For example, thelengths of 10 to 20, 10 to 25, 10 to 30, 10 to 40, 10 to 50, 10 to 60,10 to 70, 10 to 80, 10 to 90, 10 to 100, 10 to 110, 10 to 115, 10 to116, 10 to 117, 10 to 118, 10 to 119, 10 to 120, 10 to 121, 10 to 122,10 to 123, 10 to 124, 10 to 125, 10 to 126, or 10 to all amino acidswould apply to a sequence with 126 amino acids. A range of amino acidsequence lengths recited herein includes every length of amino sequencewithin the range, endpoints inclusive. The recited length of amino acidsmay start at any single position within a reference sequence whereenough amino acids follow the single position to accommodate the recitedlength. The fragment of the anti-IL-10 single domain antibody may be asubsequence of the polypeptides herein that retain at least 40% of theanti-IL-10 single domain antibody's EC₅₀ value when used in a cellularassay comprising cells that are inhibited by IL-10 to decreaseproduction of interferon gamma in the presence of ConA, which isdescribed herein in Example 9. The fragment may have 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65 70, 75, 80, 85, 90, 95, 100, 115, 116, 117,118, 19, 120, 121, 122, 123, 124, 124, or 126 amino acids. The fragmentsmay include 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 70, 75, 80,85, 90, 95, 100, 115, 116, 117, 118, 19, 120, 121, 122, 123, 124, 124,or 126 contiguous amino acids. Embodiments also include nucleic acids orpolynucleotides, encoding said amino acid sequences. A less than fulllength amino acid sequence may be selected from any portion of one ofthe sequences of SEQ ID NOS: 87-154 corresponding to the recited lengthof amino acids. A less than full length amino acid sequence may beselected from a portion of any one of SEQ ID NOS: 87-154.

The anti-IL-10 single domain antibody may increase the production ofinterferon gamma or nitric oxide when used in a cellular assaycomprising cells that are inhibited by IL-10 to decrease production ofinterferon gamma or nitric oxide.

The anti-IL-10 single domain antibody may maintain or improve the bodyweight, and, or the feed conversion ratio, of poultry fed the antibodiesrelative to poultry fed the same feed lacking the antibodies. Theanti-IL-10 single domain antibody may be dosed at less than 500 mg perkg of pelleted feed, or more preferably at less than 50 mg per kg ofpelleted feed, or even more preferably at less than 5 mg per kg ofpelleted feed, or even more preferably at less than 1 mg per kg ofpelleted feed. The anti-IL-10 single domain antibody may also improvethe body weight or feed conversion ratio of poultry when used in mash(non-pelleted) feed. The anti-IL-10 single domain antibody may maintainits affinity for IL-10 following exposure to pelleting processtemperature less than or equal to 65° C., or 70° C., or 75° C., or 80°C., or 85° C., or 90° C., or 95° C., or 100° C. The anti-IL-10 singledomain antibody may maintain its affinity for IL-10 following incubationin liquid for at least 60 seconds at a temperature less than or equal to65° C., or 70° C., or 75° C., or 80° C., or 85° C., or 90° C., or 95°C., or 100° C. The anti-IL-10 single domain antibody may have activitywhen heated to a temperature of 70° C. to 90° C. The anti-IL-10 singledomain antibody may have activity when heated to a temperature in arange between any two of the following values: 70° C., 75° C., 80° C.,85° C., or 90° C. The anti-IL-10 single domain antibody may be activefollowing exposure of a temperature of 70° C. to 90° C., or any value inbetween the foregoing values. The anti-IL-10 single domain antibody maybe an antibody stable to pepsin digestion, may have an increasedstability in the animal digestive tract, and may be produced by amicrobial host. The anti-IL-10 single domain antibody may be an antibodythat is readily degradable by pepsin. The readily degradable antibodymay completely degrade in a time period from 45 minutes to 40 minutes,from 40 minutes to 35 minutes, from 35 minutes to 30 minutes, from 30minutes to 25 minutes, from 25 minutes to 20 minutes, from 20 minutes to15 minutes, from 15 minutes to 10 minutes, from 10 minutes to 8 minutes,from 8 minutes to 6 minutes, from 6 minutes to 4 minutes, from 4 minutesto 2 minutes of the pepsin treatment. The time period for degradationmay be in a range between any two integer value between 2 minutes and 45minutes. The complete degradation of the antibody by pepsin may occur in10 minutes.

An embodiment provides one or more synthetic polynucleotides encodingthe anti-IL-10 single domain antibody or their variants describedherein. The one or more synthetic polynucleotides may comprise, consistessentially of, or consist of a sequence with at least 70, 72, 75, 80,85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to areference sequence of SEQ ID NO: 173 [chIL101A11 coding seq], SEQ ID NO:174 [chIL101A11B coding seq], SEQ ID NO: 175 [chIL101F11A coding seq],SEQ ID NO: 176 [chIL101F11B coding seq], SEQ ID NO: 177 [chIL101H1Acoding seq], or SEQ ID NO: 178 [chIL101H1B coding seq]. The one or moresynthetic polynucleotides may be included in the expression cassette tobe expressed in a host. The host may be but is not limited to amicroorganism, a plant cell, a phage, a virus, a mammalian cell, or aninsect cell.

An embodiment provides an expression cassette. The expression cassettemay comprise one or more synthetic polynucleotide encoding theantagonist IL-10R peptide, concatenated IL-10R antagonist peptides,anti-IL-10 single domain antibody or their variants described herein.

A polynucleotide sequence in an expression cassette, isolated nucleicacid, vector, or any other DNA construct herein, or utilized in a methodherein may be operably connected to one or more regulatory elements. Aregulatory element included may be a promoter. The promoter may be aconstitutive promoter which provides transcription of the polynucleotidesequences throughout the plant in most cells, tissues and organs andduring many but not necessarily all stages of development. The promotermay be an inducible promoter, which initiates transcription of thepolynucleotide sequences only when exposed to a particular chemical orenvironmental stimulus. The promoter may be specific to a host. Thepromoter may be suitable for expression of the polynucleotide in aplant, a bacterium, yeast, a mammalian cell, or an insect cell. Thepromoter may be a plant specific promoter. The promoter may be specificto a particular developmental stage, organ or tissue. A tissue specificpromoter may be capable of initiating transcription in a particularplant tissue. Plant tissue that may be targeted by a tissue specificpromoter may be but is not limited to a stem, leaves, trichomes,anthers, seed, embryo, or endosperm. A constitutive promoter herein maybe the maize Ubiquitin promoter, the rice Ubiquitin 3 promoter(OsUbi3P), the switchgrass ubiquitin promoter, the PEPC promoter, themaize Actin promoter, or the rice Actin 1 promoter. Other knownconstitutive promoters may be used, and include but are not limited toCauliflower Mosaic Virus (CAMV) 35S promoter, the Cestrum Yellow LeafCurling Virus promoter (CMP) or the CMP short version (CMPS), and theRubisco small subunit promoter.

The tissue specific promoter may include the seed-specific promoter. Theseed specific promoter may be but is not limited to the maize zeinpromoter, the rice glutelin (GluB4) promoter, the maize oleosinpromoter, or the maize globulin promoter.

The promoter may be a promoter homolog to any one of the previouslylisted promoters derived from other species, or promoter variants to thepreviously listed promoters with greater than 80% identity.

The promoter may be suitable for expressing the one or morepolynucleotides in a bacterium. The promoter may be the T7 RNApolymerase promoter, the LAC promoter or the arabinose promoter. Thepromoter may be suitable for expressing the polynucleotide in a yeast.The promoter may be the GAL promoter or the glucose promoter. Thepromoter may be any prokaryotic promoter. The prokaryotic promoter maybe a bacterial promoter, or phage promoter that is active in bacteria.The prokaryotic promoter may be any inducible promoter that is active inbacteria, or any other promoter that is active in bacteria.

Another regulatory element that may be provided is a terminatorsequence, which terminates transcription. A terminator sequence may beincluded at the 3′ end of a transcriptional unit of the expressioncassette. The terminator may be derived from a variety of genes. Theterminator may be from a eukaryote, such as a plant or mammalian cell,or a prokaryote. The terminator may be a terminator sequence from thenopaline synthase or octopine synthase genes of Agrobacteriumtumefaciens. The terminator may be maize gamma zein 27 terminator. Theterminator may be any other terminator sequence.

The one or more synthetic polynucleotide may further include one or moresignal polynucleotide sequence encoding any one of the signal peptidesdescribed herein. The expression cassette may comprise, consistessentially of, or consist of a synthetic polynucleotide sequence withat least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100% identity to a reference sequence of SEQ ID NO: 84 [xGZein27ss:chIL10sdAB1A11:KDEL], SEQ ID NO: 85 [xGZein27ss:chIL10 sdAB 1B9:KDEL],SEQ ID NO: 86 [xGZein27ss:chIL10sdAB1F11:KDEL], or SEQ ID NO: 179[xGZein27ss:chIL10sdAB1H1:KDEL].

The expression cassette including the one or more syntheticpolynucleotides may be included in a vector.

An embodiment comprises a vector containing the expression cassetteincluding one or more synthetic polynucleotides encoding the antagonistIL-10R peptide, the concatenated antagonist IL-10R peptides, or theanti-IL-10 single domain antibody of any of the above embodiments. Thevector may contain any one of the expression cassettes described in anyof the embodiments herein. The vector may be a vector used in planttransformation and that is capable of delivering its DNA into the genomeof plant cells. The vector may be a vector used for yeast and fungalexpression. The vector may be a vector for expression of the peptides,concatenated peptides or antibodies described herein used in bacterialexpression. The vector may be a vector used for mammalian or insect cellexpression.

The vector may comprise the expression cassette including syntheticpolynucleotides encoding the antagonist IL-10R peptide or concatenatedantagonist IL-10R peptides. The vector may comprise a nucleic acidsequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, or 100% identity to a reference sequence of SEQ ID NO: 69[pAG4305], SEQ ID NO: 70 [pAG4306], SEQ ID NO: 71 [pAG4308], SEQ IS NO:72 [pAG4310], SEQ ID NO: 73 [pAG4311], SEQ ID NO: 74 [pAG4312], SEQ IDNO: 75 [pAG4313], SEQ ID NO: 76 [pAG4981], SEQ ID NO: 77 [pAG4982], SEQID NO: 78 [pAG4983], and SEQ ID NO: 79 [pAG4984].

The vector may comprise the expression cassette including the syntheticpolynucleotide encoding an anti-IL-10 single domain antibody. The vectormay comprise a nucleic acid sequence with at least 70, 72, 75, 80, 85,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a referencesequence of SEQ ID NO: 155 [pAG4314], SEQ ID NO: 156 [pAG4315], SEQ IDNO: 157 [pAG4316], SEQ ID NO: 158 [pAG4317], SEQ ID NO: 159 [pAG4985],SEQ ID NO: 160 [pAG4986], SEQ ID NO: 161 [pAG4987], SEQ ID NO: 162[pAG4988], SEQ ID NO: 163 [pAG4989], SEQ ID NO: 164 [pAG4990], SEQ IDNO: 165 [pAG4991], SEQ ID NO: 166 [pAG4992], SEQ ID NO: 167 [pAG4993],SEQ ID NO: 168 [pAG4994], SEQ ID NO: 169 [pAG4995], SEQ ID NO: 170[pAG4996], SEQ ID NO: 171 [pAG4997], or SEQ ID NO: 172 [pAG4998].

An embodiment comprises a polynucleotide comprising, consistingessentially of, or consisting of a sequence that has at least 70, 72,75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identityalong its length to a contiguous portion of a polynucleotide having anyone of the sequences set forth herein or the complements thereof. Thecontiguous portion may be any length up to the entire length of asequence set forth herein or the complement thereof.

Determining percent identity of two amino acid sequences or two nucleicacid sequences may include aligning and comparing the amino acidresidues or nucleotides at corresponding positions in the two sequences.If all positions in two sequences are occupied by identical amino acidresidues or nucleotides then the sequences are said to be 100%identical. Percent identity is measured by the Smith Waterman algorithm(Smith T F, Waterman M S 1981 “Identification of Common MolecularSubsequences,” J Mol Biol 147: 195-197, which is incorporated herein byreference as if fully set forth).

In an embodiment, a transgenic plant comprising any one of syntheticpolynucleotides described herein and expressing any one of theantagonist IL-10R peptides, concatenated antagonist IL-10R peptides oranti-IL-10 single domain antibodies described herein is provided. Asused herein, the term “transgenic plants” describes plants transformedwith DNA that enables the plant containing the transformed DNA toperform a novel function; usually the transcription of the DNA,potentially at a level different from the level in wild-type plants, andpotentially the translation of the transcript into a protein, which maybe a novel protein to the plant. The transgenic plant may refer to awhole transgenic plant or tissues thereof. The tissues of transgenicplants may be any portion of a transgenic plant, including but notlimited to leaves, stems, flowers, buds, petals, grain, seed, embryo,endosperm, leaves, stalks, roots, pollen, or anthers. The tissues mayalso refer to liquid extracts made by fractionating any portion of atransgenic plant in an organic or aqueous liquid (for example,extracting protein from transgenic seeds and using the extract as asource of the transgenic protein) and using the separated liquid to feedan animal, or in animal feed, or an animal feed additive. The tissue maybe callus from a transgenic plant. The tissue may be seeds from atransgenic plant that accumulate peptides, antagonist IL-10R peptides,concatenated antagonist IL-10R peptides, or anti-IL-10 single domainantibodies described herein. A transgenic plant may be regenerated fromtissues of a transgenic plant. A transgenic plant may be a product ofsexual crossing of a first transgenic plant and a second transgenicplant or a non-transgenic plant where the product plant retains asynthetic nucleic acid introduced to the first transgenic plant. Atransgenic plant may be a product of self-pollination of a firsttransgenic plant with itself.

An embodiment provides a progeny of any one of the transgenic plantsdescribed herein. The transgenic plant may express any one of theantibodies described herein. The antibodies may target endogenousmolecules produced by the host animal ingesting the transgenic plant ortissues thereof. The targeted endogenous molecules may be but are notlimited to interleukins, cytokines, hormones, peptides, cellularreceptors, clusters of differentiation, or related molecules. Thetransgenic plants of the present disclosure may express other peptidesor proteins that impact immune response of an animal fed with thetransgenic plant or tissues thereof. The transgenic plant may contain atleast one of the expression cassettes that are described herein. Thetransgenic plant may be produced using the vectors described herein. Thetransgenic plant may be capable of producing any one of the peptides,antagonist IL-10R peptides, concatenated antagonist IL-10R peptides, oranti-IL-10 single domain antibodies described herein. The transgenicplant expressing peptides, antagonist IL-10R peptides, concatenatedantagonist IL-10R peptides, or anti-IL-10 single domain antibodiesdescribed herein, may be but is not limited to tobacco plant, cornplants, soy bean plants, or any other plant commonly eaten by animals.

The transgenic plants may express peptides and proteins that modulate,stimulate, or augment the immune system, or immune response of an animalfed the transgenic plants or tissues thereof. The transgenic plants mayexpress antibodies targeting endogenous molecules produced by the hostanimal ingesting the transgenic plants or tissues thereof. Theantibodies expressed by the transgenic plant may bind to molecules suchas interleukins, cytokines, hormones, peptides, cellular receptors,clusters of differentiation, or similar molecules. The transgenic plantsmay express other peptides or proteins that modulate various endogenousimmune system pathways, endocrine pathways, or other physiologicalsystems. More specifically, the transgenic plants may express expressone or more antibodies that bind to interleukin 10 (IL-10), or one ormore peptide or protein antagonists that interfere or block the IL-10receptor complex (IL-10R), or one or more peptide or protein moleculesthat otherwise inhibit IL-10 signaling pathways.

The transgenic plants, or tissue thereof may modulate, stimulate, oraugment the immune system, or immune response of an animal fed thetransgenic plant or tissues thereof. The transgenic plants or tissuesthereof may improve the gastrointestinal physiology of an animal eatingthe plants. The transgenic plants or tissues thereof may decrease thebinding of IL-10 with the IL-10 receptor (IL-10R) when fed to poultry.The transgenic plants or tissues thereof may maintain or improve thebody weight, and, or the feed conversion ratio, of poultry fed thetransgenic plants or tissues thereof, relative to poultry fed the samefeed lacking the transgenic plants or tissues thereof. The transgenicplants or tissues thereof may be dosed at less than 700 kg per ton ofpelleted feed, or more preferably at less than 5 kg per ton of pelletedfeed, or more preferably at less than 1 kg per ton of pelleted feed, oreven more preferably at less than 500 g per ton of pelleted feed, oreven more preferably at less than 50 g per ton of pelleted feed, or yeteven more preferably at less than 5 g per ton of pelleted feed. Thetransgenic plants and tissues thereof may also improve the body weightor feed conversion ratio of poultry when used in mash (non-pelleted)feed.

In an embodiment, a method of making any one of the transgenic plantsdescribed herein is provided. The method may comprise culturing explantsfrom a target plant and contacting them with a vector that contains atleast one expression cassette described herein. The target plant may bea corn or soy bean plant, or it may be wheat, rice, sorghum, tobacco,canola, cotton, switchgrass, or another plant. The method may includecontacting the vector with the plant explant, for example, by usingbiolistic transformation or by using Agrobacterium transformation. Oncethe explant has been contacted by the vector, methods of selecting andregenerating whole plants may be used that are known in the art.

In an embodiment, any one of the antagonist IL-10R peptides,concatenated antagonist IL-10R peptides or anti-IL-10 single domainantibodies may be isolated from the transgenic plant or plant tissue.

In an embodiment, the specific recombinant, engineered or syntheticmolecules described herein may be expressed by other hosts and may beisolated from the hosts.

In an embodiment, the transgenic plants or tissues thereof, or theisolated antagonist IL-10R peptides, concatenated antagonist IL-10Rpeptides or anti-IL-10 single domain antibodies may be included in ananimal feed.

In an embodiment, an animal feed that includes any one of the transgenicplants, or tissues thereof described herein is provided. The term“animal feed” refers to any food, feed, feed composition, preparation,additive, supplement, or mixture suitable and intended for intake byanimals for their nourishment and growth. The animal feed comprisingtransgenic plants, or plant tissues, may decrease the binding of IL-10with the IL-10R when fed to poultry. The animal feed comprisingtransgenic plants, or plant tissues, may maintain or improve the bodyweight, and, or the feed conversion ratio, of poultry fed the transgenicplants or tissues thereof, relative to poultry fed the same feed lackingthe transgenic plants or tissues thereof. The animal feed may comprisetransgenic plants or tissues thereof at less than 700 kg per ton ofpelleted feed, or more preferably at less than 5 kg per ton of pelletedfeed, or more preferably at less than 1 kg per ton of pelleted feed, oreven more preferably at less than 500 g per ton of pelleted feed, oreven more preferably at less than 50 g per ton of pelleted feed, or yeteven more preferably at less than 5 g per ton of pelleted feed. Theanimal feed or animal feed additives comprising transgenic plants andtissues thereof may also improve the body weight or feed conversionratio of poultry when used in mash (non-pelleted) feed. The animal feedmay include an isolated antagonist IL-10R peptide, concatenatedantagonist IL-10R peptides or anti-IL-10 single domain antibody. Theantagonist IL-10R peptide, concatenated antagonist IL-10R peptides oranti-IL-10 single domain antibodies included in the animal feed may beactive in the gastrointestinal environment of animals. The antagonistIL-10R peptide, concatenated antagonist IL-10R peptides or anti-IL-10single domain antibody included the animal feed may be a peptide orantibody that is stable to pepsin digestion. The antagonist IL-10Rpeptide, concatenated antagonist IL-10R peptides, anti-IL-10 singledomain antibodies included the animal feed may be a peptide or antibodythat is digested by pepsin. The animal may be a monogastric animal. Themonogastric animal may be but is not limited to a chicken, a turkey, ora duck. The antagonist IL-10R peptide, concatenated antagonist IL-10Rpeptides or anti-IL-10 single domain antibody may be active afterpreparation of the animal feed. The temperatures which feeds are exposedto during preparation may be within the range of 20° C. to 70° C.,endpoints inclusive. The temperature may be 20° C., 25° C., 30° C., 35°C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 20° C. to25° C., 20° C. to 30° C., 2° C. to 35° C., 20° C. to 40° C., 20° C. to45° C., 20° C. to 50° C., 20° C. to 55° C., 20° C. to 60° C., 20° C. to65° C., 20° C. to 70° C., 30° C. to 70° C., 40° C. to 70° C., 50° C. to70° C., 60° C. to 60° C., or less than 70° C. The temperatures whichfeeds are exposed to during pelleting may be within the range of 70° C.to 130° C., endpoints inclusive. The temperature may be 70° C., 75° C.,80° C., 85° C., 90° C., 95° C., 100° C., 105° C., 110° C., 115° C., 120°C., 125° C., 130° C., 0° C. to 75° C., 70° C. to 80° C., 70° C. to 85°C., 70° C. to 90° C., 70° C. to 95° C., 70° C. to 100° C., 70° C. to105° C., 70° C. to 110° C., 70° C. to 115° C., 70° C. to 120° C., 70° C.to 125° C., 70° C. to 130° C., 80° C. to 130° C., 90° C. to 130° C.,100° C. to 130° C., 110° C. to 130° C., 120° C. to 130° C., or less than130° C.

The antagonist IL-10R peptide, concatenated antagonist IL-10R peptidesor anti-IL-10 single domain antibody may have improved thermal stabilityand may retain activity after being exposed to high temperatures duringfeed pelleting.

In an embodiment, the animal feed may further include a feed supplement.The feed supplement may be any plant material. The plant material may bea non-transgenic plant or a transgenic plant. The plant material mayinclude a transgenic plant or a mutant plant. The plant material may bea grain that contains starch. The plant material may be a grain thatcontains fiber. The plant material may be a chemically treated forage.The plant material may be a non-transgenic plant or part thereof. Theplant material may include at least one component selected from thegroup consisting of: barley, wheat, rye, oat, corn, rice, triticale,beet, sugar beet, spinach, cabbage, quinoa, corn meal, corn pellets,corn oil, distillers grains, forage, wheat meal, wheat pellets, wheatgrain, barley grain, barley pellets, soybean meal, soybean oilcake,lupin meal, rapeseed meal, sorghum grain, sorghum pellets, rapeseed,sunflower seed, and cotton seed.

The feed supplement may be a mineral. The mineral may be a tracemineral. The mineral may be a macro mineral. The mineral may be rockphosphate or a phosphate salt. The mineral may be calcium phosphate. Thefeed supplement may be at least one vitamin. The at least one vitaminmay be a fat-soluble vitamin. The feed supplement may be an amino acid.The feed supplement may include one or more exogenous enzymes. The oneor more exogenous enzymes may include a phytase enzyme. The one or moreexogenous enzymes may include a hydrolytic enzyme. The hydrolytic enzymemay be an enzyme classified under EC3.4 as hydrolase. The hydrolyticenzymes may be, but are not limited to, xylanases, mannanases,carbohydrases, proteases, peptidases, glucanases, cellulases, lipases,phospholipases, pectinases, galactosidases, laccases, amylases,hemicellulases, or cellobiohydrolases. The enzymes may be expressed inthe transgenic plants or parts thereof included in the feed supplement.The feed supplement may include purified enzymes. The feed supplementsmay be but are not limited to growth improving additives, coloringagents, flavorings, stabilizers, limestone, stearine, starch,saccharides, fatty acids, or a gum. The coloring agents may becarotenoids. The carotenoids may be but are not limited to cantaxanthin,beta-carotene, astaxanthin, or lutein. The fatty acids may bepolyunsaturated fatty acids. The polyunsaturated fatty acids may includebut are not limited to arachidonic acid, docosohexaenoic acid (DHA),eicosapentaenoic acid (EPA) or gamma-linoleic acid.

The feed supplement may include at least one component selected from thegroup consisting of: soluble solids, fat and vermiculite, limestone,plain salt, DL-methionine, L-lysine, L-threonine, COBAN®, vitaminpremix, dicalcium phosphate, selenium premix, choline chloride, sodiumchloride, and mineral premix. The feed supplement may include fish meal,fish oil, bone meal, feather meal and animal fat. The feed supplementmay include yeast or yeast extract.

In an embodiment, a method of preparing an animal feed is provided. Themethod may include producing any one of the antagonist IL-10R peptides,concatenated antagonist IL-10R peptides or anti-IL-10 single domainantibodies described herein by any one of the methods described herein.

An embodiment provides a method of producing an animal feed. The methodmay include mixing any one of the transgenic plants or tissues thereofdescribed herein, or the progeny thereof with plant material. Thetransgenic plant may be a progeny of the transgenic plant that includeone or more synthetic polynucleotides encoding peptides and antibodiesdescribed herein. The one or more polynucleotides may be included in agenetic construct(s) or an expression cassette(s). The method maycomprise making any transgenic plant herein. The transgenic plant or itsprogeny may be the plant expressing a peptide or protein produced by themethod herein. The method may further include pelletizing the mixture.The method may further include adding a feed supplement to the mixture.The feed supplement may include at least one exogenous enzyme. The atleast one exogenous enzyme may be selected from the group consisting of:phytase, xylanase, mannanase, protease, glucanase, and cellulase.Preparing the animal feed may include combining one or more transgenicplants described herein with any other feed supplement.

An expression cassette having one or more polynucleotides encoding anantagonist IL-10R peptide, concatenated antagonist IL-10R peptides oranti-IL-10 single domain antibody in a plant may be expressed prior tothe step of step of mixing the plant, or prior to the step ofpelletizing the plant. The expression may be constitutive or theexpression may be induced. Upon the expression of the nucleic acid(s),the transgenic plant may have an increased level of the antagonistIL-10R peptide, concatenated antagonist IL-10R peptides or anti-IL-10single domain antibodies compared to the level of antagonist IL-10Rpeptide, concatenated antagonist IL-10R peptides or anti-IL-10 singledomain antibodies in a non-transgenic plant of the same geneticbackground but lacking the one or more expression cassettes.

The antagonist IL-10R peptide, concatenated antagonist IL-10R peptidesor anti-IL-10 single domain antibodies may be isolated, purified andadded to the animal feed as a pure antagonist IL-10R peptide,concatenated antagonist IL-10R peptides or anti-IL-10 single domainantibodies. The antagonist IL-10R peptide, concatenated antagonistIL-10R peptides or anti-IL-10 single domain antibodies may be isolatedfrom the intact host organism and added to the animal feed as anantagonist IL-10R peptide, concatenated antagonist IL-10R peptides oranti-IL-10 single domain antibodies composition. The antagonist IL-10Rpeptide, concatenated antagonist IL-10R peptides or anti-IL-10 singledomain antibodies may be added to the animal feed in admixture withother feed supplements. The transgenic plant including the antagonistIL-10R peptide, concatenated antagonist IL-10R antagonist peptides oranti-IL-10 single domain antibodies or the purified antagonist IL-10Rpeptide, concatenated antagonist IL-10R peptides or anti-IL-10 singledomain antibodies may be combined with other feed supplements to formpremixes.

An animal feed may be produced as mash feed. The animal feed may beproduced as pelleted feed. The milled feed stuffs may be mixed with thepremix that includes any one of the transgenic plants that include anantagonist IL-10R peptide, concatenated antagonist IL-10R peptides oranti-IL-10 single domain antibody. The antagonist IL-10R peptide,concatenated antagonist IL-10R peptides or anti-IL-10 single domainantibody may be an antagonist IL-10R peptide, concatenated antagonistIL-10R peptides or anti-IL-10 single domain antibody that is/are stableto pepsin digestion. The antagonist IL-10R peptide, concatenatedantagonist IL-10R peptides or anti-IL-10 single domain antibody may bean antagonist IL-10R peptide, concatenated antagonist IL-10R peptides oranti-IL-10 single domain antibody that is/are digestible by pepsin. Themilled feed stuffs may include the plant material and the feedsupplements described herein. The feed supplements may include one ormore exogenous enzymes described herein. Enzymes may be added as liquidor solid formulations. For mash feed, a solid or liquid peptideformulation may be added before or during the mixing step. For pelletedfeed, the peptide preparation may be added before or after the pelletingstep. The antagonist IL-10R peptide, concatenated antagonist IL-10Rpeptides or anti-IL-10 single domain antibody may be included in premix.The premix may also include vitamins and trace minerals. Macro mineralsmay be added separately to animal feedstock.

An embodiment comprises a method of treating or preventing agastrointestinal infection in an animal. The gastrointestinal infectionmay be caused by a gastrointestinal pathogen. As used herein, agastrointestinal pathogen may include a bacterium, yeast, fungi,archaea, virus, protozoa, or other infectious agent that is capable ofreplication inside or outside of the infected host animal, and causesirritation, necrosis, cellular disruption, or cellular damage within theinfected host animal, or otherwise stimulates or modulates the immunesystem of the infected host animal. The gastrointestinal pathogen maybelong to the genus Eimeria. The gastrointestinal pathogen may be but isnot limited to Eimeria tenella, Eimeria acervulina, or Eimeria maxima.The method may include administering to an animal any one of theantagonist IL-10R peptides or anti-IL-10 single domain antibodiesdescribed herein. Administering may be performed by any known route, forexample, by injection. Administering may be performed by feeding theinfected animal with the transgenic plant expressing one or moreantibodies that bind to IL-10, or one or more peptide or proteinantagonists to the IL-10R, or plant tissues thereof, or animal feed orfeed compositions containing the transgenic plants and tissues thereof.The method comprises administering any one of the antagonist IL-10Rpeptides or anti-IL-10 single domain antibodies described herein in atherapeutically effective amount. As used herein, a therapeuticallyeffective amount of an antagonist IL-10 peptide, concatenated antagonistIL-10 peptides or anti-IL-10 single domain antibody is an amounteffective to reduce the symptoms of the gastrointestinal disease in theanimal when administered daily for a period of from one week to twomonths. The therapeutically effective amounts of the antagonist IL-10Rpeptide or concatenated antagonist IL-10R peptides may be a dose of lessthan 500 mg per kg of pelleted feed, or more preferably less than 50 mgper kg of pelleted feed, or even more preferably less than 5 mg per kgof pelleted feed, or even more preferably less than 1 mg per kg ofpelleted feed.

The therapeutically effective amounts of the anti-IL-10 single domainantibody may be at a dose of less than 500 mg per kg of pelleted feed,or more preferably at a dose of less than 50 mg per kg of pelleted feed,or even more preferably at a dose of less than 5 mg per kg of pelletedfeed, or even more preferably at a dose of less than 1 mg per kg ofpelleted feed.

The therapeutically effective amounts of the transgenic plants ortissues thereof may be at a dose of less than 700 kg per ton of pelletedfeed, or more preferably at a dose of less than 5 kg per ton of pelletedfeed, or more preferably at a dose of less than 1 kg per ton of pelletedfeed, or even more preferably at a dose of less than 500 g per ton ofpelleted feed, or even more preferably at a dose of less than 50 g perton of pelleted feed, or yet even more preferably at a dose less than 5g per ton of pelleted feed.

The therapeutically effective amounts of the animal feed may comprisetransgenic plants or tissues thereof at less than 700 kg per ton ofpelleted feed, or more preferably at less than 5 kg per ton of pelletedfeed, or more preferably at less than 1 kg per ton of pelleted feed, oreven more preferably at less than 500 g per ton of pelleted feed, oreven more preferably at less than 50 g per ton of pelleted feed, or yeteven more preferably at less than 5 g per ton of pelleted feed.

An embodiment comprises a method of stimulating or modulating the immunesystem and improving gastrointestinal physiology of an animal comprisingfeeding the animal with the transgenic plants or tissues thereof. Asused herein, the term “modulate” means to change, or respond to astimulus. In this context “modulate” could mean to increase a responseor decrease a response. With regards to modulating an immune response,it means to stimulate or to decrease an immune response. Words that areused synonymously with decrease as it relates to modulation of aresponse include blocking, interfering, antagonizing, lowering,alleviating, shutting down, or removing. The term gastrointestinalphysiology describes the biological state of an animal'sgastrointestinal tract, including the foregut, midgut, and hindgut. Theactual anatomical features of the gastrointestinal tract may vary amonganimal species, but in poultry include the esophagus, crop,proventriculus, ventriculous, gizzard, duodenum, jejunum, ileum, smallintestine, large intestine, cloaca, and ceca. The biological state ofthe gastrointestinal tract may be described as healthy or normal,lacking any abnormal visual or pathological observation, or aberranthistological evaluation. The biological state of the gastrointestinaltract may be described as inflamed, infected, or necrotic, all of whichdescribe a physiological state that is impaired and could be improved toa normal or healthy state.

In an embodiment, a method of improving the gastrointestinal physiologyof an animal is provided. The method may comprise feeding the animal anyof the transgenic plants, or plant parts, described herein. In anembodiment, the method may comprise feeding the animal any of theanti-IL-10 single domain antibodies, peptides, or antagonist IL-10R sdescribed herein.

In an embodiment, a method of improving animal performance or animalgastrointestinal physiology is provided. The method may comprise feedingthe animals any of the transgenic plants expressing one or moreantibodies that bind to IL-10, or one or more peptide or proteinantagonists to the IL-10R, or plant tissues thereof, or feed, or feedcompositions containing the transgenic plants or tissues thereof, oranti-IL-10 single domain antibodies, or the IL-10R antagonists. Themethod may comprise feeding the animals any one of the transgenicplants, or plant parts described herein. As used herein, animalperformance is synonymous with animal growth or animal productivity, andeach term can be used interchangeably. Animal performance relates to theweight gain of the animal over time, and to the animal's feed conversionratio, which is defined as the mass of feed eaten by the animal dividedby the weight gain of the animal. These terms may be used to describeeither, or both, weight gain and feed conversion ratio, so animprovement in animal performance may indicate an increase in weightgain relative to control animals, and, or, a decrease (less feed eatenper mass of animal growth) in feed conversion ratio. In an embodiment,the method may comprise feeding an animal any of the animal feed oranimal feed additives comprising any of the anti-IL-10 single domainantibodies, peptides, IL-10R antagonists, or transgenic plants ortissues thereof, described herein.

The following list includes particular embodiments of the presentinvention. But the list is not limiting and does not exclude alternateembodiments, or embodiments otherwise described herein. Percent identitydescribed in the following embodiments list refers to the identity ofthe recited sequence along the entire length of the reference sequence.

EMBODIMENTS

1. At least one antagonist IL-10R peptide, wherein (i) the at least oneantagonist IL-10R peptide is one peptide comprising an amino acidsequence with at least 90% identity to a reference sequence selectedfrom the group consisting of: SEQ ID NOS: 1-13, or (ii) the at least oneantagonist IL-10R peptide comprises concatenated peptides comprising anamino acid sequence with at least 90% identity to a reference sequenceselected from the group consisting of: SEQ ID NOS: 32-40.

2. The at least one antagonist IL-10R peptide of embodiment 1, whereineach of the concatenated peptides are linked to each other by one ormore linkers.

3. The at least one antagonist peptide of any one or both of embodiments1 and 2, wherein the one or more linkers comprise a sequence selectedfrom the group consisting of SEQ ID NOS: 41-44, and 65.

4. The at least one antagonist IL-10R peptide of any one or more ofembodiments 1-3, wherein the at least one peptide or each one of theconcatenated peptides comprise an N-terminal signal peptide orC-terminal signal peptide, or both.

5. The at least one antagonist IL-10R peptide of any one or more ofembodiments 1-4, wherein the N-terminal signal peptide is selected froma group consisting of: OsGluB4sp (rice Glu-B4 glutelin signal peptide),BAASS (barley alpha amylase signal sequence), PR1 (pathogenesis relatedprotein), or zein 27 (xGZm27ss) signal peptide.

6. The at least one antagonist IL-10R peptide of any one or more ofembodiments 1-5, wherein the peptide is stable at a temperature in arange from 70° C. to 90° C.

7. The at least one antagonist IL-10R peptide of any one or more ofembodiments 1-6, wherein the peptide is digestible by pepsin.

8. The at least one antagonist IL-10R peptide of any one or more ofembodiments 1-6, wherein the peptide is stable to digestion by pepsin.

9. The at least one antagonist IL-10R peptide of any one or more ofembodiments 1-8, wherein the C-terminal signal peptide is selected froma group consisting of: KDEL (SEQ ID NO: 29), HDEL (SEQ ID NO: 30),SEKDEL (SEQ ID NO: 31), HvVSD from barley polyamine oxidase, or HvAlefrom barley aleurone (thiol protease).

10. A synthetic polynucleotide encoding the at least one antagonistIL-10R peptide of any one or more of embodiments 1-9.

11. The synthetic polynucleotide of embodiment 10, wherein the syntheticpolynucleotide comprises a sequence with at least 90% identity to areference sequence selected from the group consisting of: 16-28, and 56.

12. An anti-IL-10 single domain antibody that binds to a polypeptidecomprising an amino acid sequence of SEQ ID NO: 80.

13. The anti-IL-10 single domain antibody of embodiment 12, wherein theantibody has a molecular mass in a range of 10 kDa to 20 kDa.

14. The anti-IL-10 single domain antibody of any one or both ofembodiments 12 and 13, wherein the antibody is stable at a temperaturein a range from 70° C. to 90° C.

15. The anti-IL-10 single domain antibody of any one or more ofembodiments 12-14, wherein the antibody binds to chicken I1-10 with anEC₅₀ of 30 nM or less in a cell ELISA assay.

16. The anti-IL-10 single domain antibody of any one or more ofembodiments 12-15, wherein the antibody comprises an amino acid sequencewith at least 90% identity to a sequence selected from the groupconsisting of SEQ ID NOs: 87-154.

17. The anti-IL-10 single domain antibody of any one or more ofembodiments 12-16, wherein the antibody comprises the amino acidsequence with at least 90% identity to a sequence selected from thegroup consisting of SEQ ID NOs: 89, 135, and 146.

18. The anti-IL-10 single domain antibody of any one or more ofembodiments 12-17, wherein the antibody is fused to an N-terminal signalpeptide or C-terminal signal peptide, or both.

19. The anti-IL-10 single domain antibody of any one or more ofembodiments 12-18, wherein the N-terminal signal peptide is selectedfrom a group consisting of: OsGluB4sp (rice Glu-B4 glutelin signalpeptide), BAASS (barley alpha amylase signal sequence), PR1(pathogenesis related protein), or zein 27 (xGZm27ss) signal peptide.

20. The anti-IL-10 single domain antibody of any one or more ofembodiments 12-19, wherein the C-terminal signal peptide is selectedfrom a group consisting of: KDEL (SEQ ID NO: 29), HDEL (SEQ ID NO: 30),SEKDEL (SEQ ID NO: 31), HvVSD from barley polyamine oxidase, or HvAlefrom barley aleurone (thiol protease).

21. The anti-IL-10 single domain antibody of any one or more ofembodiments 12-20, wherein the anti-IL-10 single domain antibody isdigestible by pepsin.

22. The anti-IL-10 single domain antibody of any one or more ofembodiments 12-20, wherein the anti-IL-10 single domain antibody isstable to digestion by pepsin.

23. The anti-IL-10 single domain antibody of any one or more ofembodiments 12-22, wherein the anti-IL-10 single domain antibody isstable to a temperature exposure of greater than 70° C. and less than100° C.

24. A synthetic polynucleotide encoding the anti-IL-10 single domainantibody of any one or more of embodiments 12-23.

25. The synthetic polynucleotide of embodiment 24, wherein the syntheticpolynucleotide comprises a sequence with at least 90% identity to areference sequence selected from the group consisting of: SEQ ID NOs:173-178.

26. A transgenic plant or tissues thereof comprising one or morepolynucleotides encoding the at least one antagonist IL-10R peptide ofany one or more of embodiments 1-9, or the anti-IL-10 single domainantibody of any one or more of embodiments 12-23.

27. A transgenic plant or tissues thereof comprising one or morepolynucleotides encoding the at least one antagonist IL-10R peptide, orthe anti-IL-10 single domain antibody.

28. The transgenic plant or tissues thereof of embodiment 27, whereinthe antagonist IL-10R peptide is one peptide comprising an amino acidsequence with at least 90% identity to a reference sequence selectedfrom the group consisting of: SEQ ID NOS: 1-13.

29. The transgenic plant or tissues thereof of any one or bothembodiments 27 and 28, wherein the antagonist IL-10R peptide comprisesconcatenated antagonist IL-10R peptides comprising an amino acid with atleast 90% identity to a reference sequence selected from the groupconsisting of SEQ ID NOS: 32-40.

30. The transgenic plant or tissues thereof of any one or more ofembodiments 27-29, wherein the anti-IL-10 single domain antibody bindsto a polypeptide comprising an amino acid sequence of SEQ ID NO: 80.

31. The transgenic plant or tissues thereof of any one or more ofembodiments 27-30, wherein the anti-IL-10 single domain antibody has amolecular mass in a range of 10 kDa to 20 kDa.

32. The transgenic plant or tissues thereof of any one or more ofembodiments 27-31, wherein the anti-IL-10 single domain antibody isstable at a temperature in a range from 70° C. to 90° C.

33. The transgenic plant or tissues thereof of any one or more ofembodiments 27-32, wherein the anti-IL-10 single domain antibody bindsto chicken I1-10 with an EC50 of 30 nM or less in a cell ELISA assay.

34. The transgenic plant or tissues thereof of any one or more ofembodiments 27-33, wherein the anti-IL-10 single domain antibodycomprises an amino acid sequence with at least 90% identity to asequence selected from the group consisting of SEQ ID NOs: 87-154.

35. The transgenic plant or tissues thereof of any one or more ofembodiments 27-34, wherein the anti-IL-10 single domain antibodycomprises the amino acid sequence with at least 90% identity to asequence selected from the group consisting of SEQ ID NOs: 89, 135, and146.

36. The transgenic plant or tissues thereof of any one or more ofembodiments 27-35, wherein the one or more polynucleotides comprise asequence with at least 90% identity to a reference sequence selectedfrom the group consisting of SEQ ID NOS: 16-28, and 56.

37. The transgenic plant or tissues thereof of any one or more ofembodiments 27-36, wherein the one or more polynucleotides comprise asequence with at least 90% identity to a reference sequence selectedfrom the group consisting of SEQ ID NOS: 173-178.

38. The transgenic plant or tissues thereof of any one or more ofembodiments 27-37, wherein the anti-IL-10 single domain antibody isdigestible by pepsin.

39. The transgenic plant or tissues thereof of any one or more ofembodiments 27-37, wherein the anti-IL-10 single domain antibody isstable to digestion by pepsin.

40. The transgenic plant or tissues thereof of any one or more ofembodiments 27-39, wherein a plant is selected from the group consistingof: corn, soybean, wheat, rice, sorghum, canola, cotton, andswitchgrass.

41. An animal feed comprising the transgenic plant or tissues thereof ofany one or more of embodiments 26-40.

42. An animal feed comprising at least one antagonist IL-10R peptide ofany one or more of embodiments 1-9, or an anti-IL-10 single domainantibody of any one or more of embodiments 12-23.

43. The animal feed of embodiment 42, wherein the at least one IL-10Rantagonist IL-10R peptide, or the anti-IL-10 single domain antibody isactive upon expression in the plant and exposure to a temperature in therange from 25° C. to 130° C.

44. The animal feed of any one or more of embodiments 41, or 42-43further comprising a feed supplement.

45. The animal feed of any one or more of embodiments 41, or 42-44,wherein the feed supplement is plant material.

46. The animal feed of any one or more of embodiments 41, or 42-45,wherein the plant material is a non-transgenic plant or a transgenicplant.

47. The animal feed of any one or more of embodiments 41, or 42-46,wherein the plant material includes at least one component selected fromthe group consisting of: corn meal, corn pellets, wheat meal, wheatpellets, wheat grain, barley grain, barley pellets, soybean meal,soybean oilcake, sorghum grain and sorghum pellets.

48. The animal feed of any one or more of embodiments 41, or 42-47,wherein the feed supplement includes one or more exogenous enzymes.

49. The animal feed of any or more of embodiments 41, or 42-48, whereinthe one or more exogenous enzymes includes a hydrolytic enzyme selectedfrom the group consisting of: xylanase, endoglucanase, cellulase,protease, phytase, amylase and mannanase.

50. The animal feed of any one or more of embodiments 41, or 42-49,wherein the feed supplement includes at least one component selectedfrom the group consisting of: soluble solids, fat and vermiculite,limestone, plain salt, DL-methionine, L-lysine, L-threonine, COBAN®,vitamin premix, dicalcium phosphate, selenium premix, choline chloride,sodium chloride, and mineral premix.

51. A method of treating or preventing a gastrointestinal infection inan animal comprising feeding the animal the at least one antagonistIL-10R peptide of any one or more of embodiments 1-9, the anti-IL-10single domain antibody of any or more of embodiments 12-23, thetransgenic plant or tissues thereof of any one or more of embodiments26-40, or the animal feed of any one more of embodiments 41-50.

52. The method of embodiment 51, wherein the gastrointestinal infectionis caused by a gastrointestinal pathogen selected from the groupconsisting of: bacteria, yeast, fungi, archae, virus, and protozoa.

53. The method of any one or both of embodiments 51 and 52, wherein thegastrointestinal pathogen belongs to the genus Eimeria.

54. The method any one or more of embodiments 51-53, wherein thegastrointestinal pathogen is selected from the group consisting of:Eimeria tenella, Eimeria acervulina, and Eimeria maxima.

55. A method of stimulating or modulating the immune system andimproving gastrointestinal physiology of an animal comprising feedingthe animal the at least one antagonist IL-10R peptide of any one or moreof embodiments 1-9, the anti-IL-10 single domain antibody of any or moreof embodiments 12-23, the transgenic plant or tissues thereof of any oneor more of embodiments 26-40, or the animal feed of any one or more ofembodiments 41-50.

56. A method of improving animal performance comprising feeding ananimal the at least one antagonist IL-10R peptide of any one or more ofembodiments 1-9, the anti-IL-10 single domain antibody of any one ormore of embodiments 12-23, the transgenic plant or tissues thereof ofany one or more of embodiments 26-40, or the animal feed of any one ormore of embodiments 41-50.

57. A method of preparing an animal feed comprising mixing theantagonist IL-10R peptide of any one or more of embodiments 1-9, theanti-IL-10 single domain antibody of any one or more of embodiments12-23, or the transgenic plant or tissues thereof of any one or more ofembodiments 26-40 with plant material to form a mixture.

58. The method of embodiment 57, wherein the method further comprisespelletizing the mixture.

59. The method of any one or both of embodiments 57 and 58, wherein themethod further comprises adding a feed supplement to the mixture.

60. The method of any one or more of embodiments 57-59, wherein theplant material is a non-transgenic plant or a transgenic plant.

61. The method of any one or more of embodiments 57-60, wherein theplant material includes at least one component selected from the groupconsisting of: corn meal, corn pellets, wheat meal, wheat pellets, wheatgrain, barley grain, barley pellets, soybean meal, soybean oilcake,sorghum grain and sorghum pellets.

62. The method of any one or more of embodiments 57-61, wherein the feedsupplement includes one or more exogenous enzymes.

63. The method of any or more of embodiments 57-62, wherein the one ormore exogenous enzymes includes a hydrolytic enzyme selected from thegroup consisting of: xylanase, endoglucanase, cellulase, protease,phytase, amylase and mannanase.

64. The method of any one or more of embodiments 57-63, wherein the feedsupplement includes at least one component selected from the groupconsisting of: soluble solids, fat and vermiculite, limestone, plainsalt, DL-methionine, L-lysine, L-threonine, COBAN®, vitamin premix,dicalcium phosphate, selenium premix, choline chloride, sodium chloride,and mineral premix.

Further embodiments herein may be formed by supplementing an embodimentwith one or more elements from any one or more other embodiments herein,and/or substituting one or more elements from one embodiment with one ormore elements from one or more other embodiments

EXAMPLES

The following non-limiting examples are provided to illustrateparticular embodiments. The embodiments throughout may be supplementedwith one or more details from one or more examples below, and/or one ormore elements from an embodiment may be substituted with one or moredetails from one or more examples below.

Example 1. Strategies for Engineering Peptides and Antibodies

While reducing IL-10 levels prior to and during Eimeria infection canhelp control the negative effects of Coccidiosis, the mechanisms knownin the art that have been employed to reduce IL-10 levels are expensiveand have questionable robustness to be employed broadly in industry. Theshortcomings of the existing technologies (using isolated, or partiallypurified, peptides or antibodies) to control Coccidiosis can beaddressed in several important ways using biotechnology to design novelproducts that target the IL-10 signaling pathway. First, by broadeningantibody discovery and development beyond the common target chickenproduction system, as is currently done by inoculating maternal hens oreggs with conjugated IL-10 peptides, novel antibodies and peptides canbe developed that have been specifically tailored to controllingCoccidiosis. The peptides and synthetic antibodies developed herein wereengineered to have high affinity (thus reducing dosing levels), improvedthermal stability (to survive pelleting when mixed into animal feed),and low molecular weight to promote high-levels of expression (tomaximize production economics), properties that are not found innaturally occurring peptides and antibodies and that could not be simplyselected for in nature, nor could these properties be efficientlyreplicated in a hen or egg production system without undueexperimentation. Second, by engineering the genes encoding the peptidesand antibodies developed herein into plants, their delivery can be madeby directly feeding the plants or plant tissues without additionalisolation, or purification, or formulation, into the diet. This greatlybenefits production and animal administration economics. That such acombination of technologies works effectively in controlling Coccidiosiswas unexpected. It was anticipated that antibodies and peptidesdelivered in whole grain or meal, with no isolation, would either notsurvive the pelleting process when being mixed in feed (which is oftenthe case when using larger antibodies, such as IgG's or IgY's, andpeptides), not diffuse adequately from the plant matrix and be readilyavailable to the animal at sufficient concentrations to modulate theIL-10 signaling pathway, or would be rapidly degraded in the digestivetract. Unexpectedly, the combination of technologies used to make theproducts described herein, was able to overcome these challenges andaddress the challenges confronting current methods used in the art forcontrolling Coccidiosis.

Plant expression of heterologous peptides and proteins is one of theleast expensive recombinant protein production systems on the planet. Byengineering corn or soy beans to produce anti-IL-10 antibodies, IL-10Rpeptide antagonists, or other molecules that inhibit IL-10 signaling,these molecules can be made at high concentrations, e.g., aconcentration in a range from 0.01 mg of heterologous protein(anti-IL-10 antibodies, IL-10R peptide antagonists, or other proteins orpeptides that inhibit IL-10 signaling) per gram of milled grain up to asmuch as 20 mg of heterologous protein per gram of milled grain.Heterologous proteins and peptides produced in seed tissue are naturallystabilized in the seed as it progresses through its desiccation processfollowing seed development. Antibodies and peptide antagonists producedin corn or soy beans can be delivered directly into poultry diets bysimply milling the grain and mixing it with the other ingredients. Whileother processing and formulation steps may be used, there is no need foradditional processing steps that would be required if these productswere produced by fermentation or by inoculating eggs, both of which aremore expensive processes. Further, because these molecules are encodedby DNA that is stably integrated into the plants' genome, there is theopportunity to further engineer these molecules and endow them withbeneficial properties that cannot be implemented when generatingantibodies in maternal hens or by inoculating eggs.

Engineering of antibodies, proteins, and peptides provides additionalopportunities to address industry needs in modulating IL-10 signalingpathways. Anti-IL-10 antibodies can be generated by any number of hosts,including camels, calves, chickens, goats, horses, humans, llamas, mice,rabbits, rats, sharks, and many other species. The particular choice ofhost for generating an antibody may depend on a variety ofconsiderations including the choice of antigen (will the antigen berecognized as self by the host or recognized as foreign and generate anappropriate immune response), the choice of antibody (IgG, IgY, apolyclonal, monoclonal, etc.), ease of working with the host, the amountof antibody desired, the intended species the antibody will be used in,and the type of antibody desired (full length (approximately 120-160kDa), antibody fragments (Fab that can be approximately 50 kDa, or scFvthat can be approximately 25 kDa), or single domain antibodies (that canbe approximately 10-20 kDa)). Single domain antibodies are smallsynthetic antibodies abbreviated by sdAb, sdAB, VHH, or VNAR, and havespecific features that make them suitable for targeting IL-10 whendelivered through feed into an animal's digestive tract. In particular,sdAbs are generally recognized as having improved thermal stabilityrelative to full length and other antibody fragments, and are verysusceptible to molecular engineering, which provides the possibility offurther improving their thermal stability and affinity, both of whichmay help in reducing the necessary dose required in the animal feed tomodulate the IL-10 signaling pathway (E. R. Goldman, G. P. Anderson, J.L. Liu, J. B. Delehanty, L. J. Sherwood, L. E. Osborn, L. B. Cummins,and A. Hayhurst, 2006, Facile Generation of Heat-Stable Antiviral andAntitoxin Single Domain Antibodies from a Semisynthetic Llama Library,Annal. Chem., 78:8245-8255, which is incorporated herein by reference asif fully set forth). Chicken IL-10 (amino acid residues in positions 2to 151 of SEQ ID NO: 80) was compared with llama IL-10 (SEQ ID NO: 207).Sequence alignment analysis showed the sequences to be only 48%identical. Based on this analysis, the full-length Gallus gallus IL-10(chicken IL-10 or cIL-10) was selected as the target antigen forgenerating sdAbs in camels or llamas. Because llamas are not exposed tocIL-10, an endogenous chicken interleukin, and it's only through abiotechnology process that cIL-10 can be made, isolated and dosed into allama, llamas are naive to cIL-10 and it was found that it's possible togenerate a significant immune response as shown in FIG. 3. Further,given that IL-10 is known to dimerize in vivo (K. Asadullah, W. Sterry,H. D. Volk, “Interleukin-10 Theraphy—Review of a New Approach”,Pharmacological Reviews, 55(2):241-269, 2003), using IL-10 peptides astarget antigens is challenging because peptide epitopes may be selectedthat are not normally exposed in the IL-10 dimer in vivo. Thus, librarygeneration and antibody screening may be more readily optimized, andsynthetic antibodies developed, using full-length cIL-10. Anotheradvantage of developing a single molecule for expression is that it canbe selected from a diversity of molecules made by the inoculated host,allowing for screening and selection of a highly optimized sdAb that canbe reproducibly made with great efficiency in corn or another host. Inthis way, many of the current challenges with modulating the IL-10pathway in poultry can be addressed to bring this innovation into themarketplace.

Example 2. Peptide Selection

Synthetic peptides were designed by analyzing the crystal structures ofhuman IL-10/IL-10R1 complexes, determining portions of the sequences ofthe human proteins that contribute to binding, and finding the analogoussequences in the chicken proteins by aligning the sequences of human andchicken IL-10 and IL-10R1, respectively, with Clustal Omega (Diaz-Valdezet al., 2011, Josephson et al., 2001, Naiyer et al., 2013, Ni et al.,2016, Reineke et al., 1998, Yoon et al., 2005, Zdanov et al., 1996).Amino acid sequences were obtained from Pub Med; and the presumed signalpeptide, transmembrane, and intracellular sequences were removed fromthe alignments. All peptide sequences were checked for known allergenicepitopes using the Allergen Online Database.

Human IL-10 residues involved in binding to its receptor as determinedby peptide mapping are as follows:

Chicken ------ LEPTCLHFSELLPARLRELRVKFEEIKDYFQSRDD HumaSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQ.   .   * :* **   **  **:**  *..:* :** :*: ChickenELNIQLLSSELLDEFKGTFGCQSVSEMLRFYTDEVLPRAMQTSTSHQQSMGDLGNMLLGL Human

ChickenKATMRRCHRFFTCEKRSKAIKQIKETFEKMDENGIYKAMGEFDIFINYIEEYLLMRRR- (SEQ ID NO: 80)Human

Human (AAA63207.1; SEQ ID NO: 81) and chicken (NP_001004414.2; SEQ IDNO: 80) sequences were obtained and edited as described above. HumanIL-10 residues that are involved in binding to human IL-10R1 are inboldface and colored gray (Reineke et. al., 1998). Chicken IL-10sequence alignment with the human IL-10 sequence is also shown.

Human IL 40 residues that bury >5 Å² surface area in the complex withthe receptor (Yoon, et, al., 2005).

Chicken ------ LEPTCLHFSELLPARLRELRVKFEEIKDYFQSRDD Human

Chicken ELNIQLLSSELLDEFKGTFGCQSVSEMLRFYTDEVLPRAMQTSTSHQQSMGDLGNMLLGLHuman

ChickenKATMRRCHRFFTCEKRSKAIKQIKETFEKMDENGIYKAMGEFDIFINYIEEYLLMRRR- (SEQ ID NO: 80)Human

Chicken IL-10 sequence (SEQ ID NO: 80) alignment with the human IL-10sequence (SEQ ID NO: 81) is shown, with the two segments of the humanIL-10 sequence that contribute the majority of binding surface(Diaz-Valdez et al., 2011; Josephson et al., 2001; Naiyer et al., 2013;Ni et al., 2016; Reineke et al., 1998; Yoon et al., 2005; and Zdanov etal., 1996), are indicated in boldface and colored gray. Helical regionsare underlined.

Examples of peptide design based on alignment with predicted sequenceregions of binding interactions:

Chicken Human

Chicken

Human -LDNLLL KESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTL *: **...**::*** :***::***::** :**:*:* : . . :  :..**: *  * ChickenKATMRRCHRFFTCEKRSKAIKQIKETFEKMDENGIYKAMGEFDIFINYIEEYLLMRRR- (SEQ ID NO: 80)Human RLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 81):   : ** *** *:  ** : : * ** : : * : * : : * : * : : * : * *** **. ** ** *** ** * * :  *:  *

Binding hot spots and helical regions of human IL-10 are designated asshown previously. Sequences within the chicken IL-10 sequence thatencompass peptides P25 (SEQ ID NO: 5) and P26 (SEQ ID NO: 6) aredesignated by boldface italics and colored gray. Peptide P21 (SEQ IDNO: 1) consists of P25, P26, and all of the chicken IL-10 residuesbetween them in the sequence.

Examples of peptide design based on alignment with binding hot spots:

Chicken ------ LEPTCLHFSELLPARLRELRVKFEEIKDYFQSRDD HumanSPGQGTQSENSCTHFPGNLP NMLRDLRDAFSRVKTFFQ MKDQ.    .    *  : *  **    **   ** * *   * . . : *  : * * : * : ChickenELNIQLLSSELLDEFKGTFGCQSVSEMLRFYTDEVLPRAMQTSTSHQQSMGDLGNMLLGL Human-LDNLLL KESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTL *:  **...**::*** :***::***::** :**:*:* : . . :  :..**: *  * Chicken

Human RLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFNYIEAYMTMKIRN (SEQ ID NO: 81):   : ** *** *:  ** : : * ** : : * : * : : * : * : : * : * *** **. ** ** *** ** * * :  *:  *

Binding hot spots and helical regions of human IL-10 are designated aspreviously shown. Peptide P27 (SEQ ID NO: 7) is designated by boldface,italicized and is colored in gray. Peptide P22 (SEQ ID NO: 2) isdesignated in boldface and included in a frame. Peptide 27 is the regionwithin the P22 sequence.

Alignment of human (SEQ ID NO: 83) and chicken (SEQ ID NO: 82) IL-10R1soluble domain sequences:

Human IL-10R1HGTELPSPPSVWFEAEFFHHILHWTPIPNQSESTCYEVALLRYGIE-SWNSISNCSQTLSChicken IL-10r1--ELRLKPTRVRFVAEMVYHLLQWEPGPDAPSDTRYDVEHKIYGTNSPWTAIPNCMKIHG      .*  * * **:.:*:*:* *  :  ..* *:*    ** :  *.:* ** :  .Human IL-10R1YDLTAVTLDLYHSNGYRARVRAVDGSRHSNWTVTNTRFSVDEVTLTVGSVNLEIHNGFILChicken IL-10R1HSCDLTYYTLDPSLRYYARVRAVVGNHTSDWKRTNA-FSPQEASLRLSGHSLAVTDNSIH:.   .   *  *  * ****** *.: *:*. **: ** :*.:* :.. .* : :. *Human IL-10R1GKIQLPRPKMAPANDTYESIFSHFREYEIAIRKVPGNFTFTHKKVKHENFSLLTSGEVGEChicken IL-10R1VQLQLLL-RAGNRTVKYDDIQKHARRYRVYIRRARDNQTYEVWETAS-EFYIRNLFWNTE ::**   : .  . .*:.* .* *.*.: **:. .* *:   :.   :* : .     *Human IL-10R1FCVQVKPSVASRSNKGMWSKEECISLTRQYFTVTN---VIIFFAFVLL---LSGALAYCL (SEQ ID NO: 83)Chicken IL-10R1 YCISVEPDVASRHIPAMRTAEQCVTIGHRDESAEL (SEQ ID NO: 82):*:.*:*.****   .* : *:*::: ::  :.

Human (NP_001549.2) and chicken (NP_001034686.1) sequences were obtainedand edited as described above. Human IL-10R1 residues that are involvedin binding to human IL-10 (FIG. 5 of Reineke et. al., 1998) aredesignated by boldface. Chicken IL-10R1 sequence alignment with thehuman IL-10R1 sequence is also shown; intracellular and transmembranedomains are omitted for clarity.

Example 3. Peptide Screening

Peptides were chemically synthesized, desalted, and purified to >98%purity by Watsonbio, Inc., Houston Tex. Peptide stock solutions wereprepared at 10 mM in DMSO and diluted in assay buffer to 0.05 mM.Peptides were screened at Marin Biologic Laboratories, Inc., Novato,Calif., by determining their effectiveness at blocking the inhibitoryeffect of chicken IL-10 on the induction of interferon gamma (IFN-γ)induced by concanavalin A (ConA) (or alternatively phytohemagglutinin(PHA) may be used) in chicken spleen cells (Wu et al. (2016) andRothwell et al. (2004)). Briefly, lymphocytes and mononuclear cells wereisolated from chicken spleens by differential centrifugation onFicoll-Hypaque. Cells were cultured at 5×10⁶ cells/mL in wells of a96-well plate for 72 hours in the presence of peptide, 1.2 μg/mL ConA(or alternatively 12.5 μg/mL PHA), and with or without cIL-10. Spleencells were also incubated without peptides and/or without ConA (or PHA)as controls. Levels of IFN-γ in the supernatants were determined byELISA.

The use of peptides derived from IL-10s of several species that can beadded to feed to reduce respiratory and intestinal illness in theseanimals is known in the art. See U.S. Pat. No. 8,652,457 B2 and U.S.patent application publication No. US2016/0280778 A1, which areincorporated by reference herein as if fully set forth. These peptideswere designed to elicit an immune response in the animals, which wouldlead to the production of anti-IL-10 antibodies. In contrast, peptidesdescribed herein were designed with the goal of directly interferingwith binding of chicken IL-10 to its receptor (that is, as IL-10Rantagonists). As a result, the peptides described here were designed inpart to mimic sections of either cIL-10 or the R1 subunit of the cIL-10receptor and to compete with either cIL-10 or cIL-10R for binding to theother species, as opposed to the peptides in the previous patents thatwere designed to incorporate antigenic features of IL-10.

TABLE 1 Amino acid sequences of peptides SEQ ID NO Description Sequence1 P21 PARLRELRVKFEEIKDYFQSRDDELNIQLLSSEL LDEFKG 2 P22ENGIYKAMGEFDIFINYIEEYLLMRRR 3 P23 PARLRELRVKFEEIKDYFQGGGSGGGSQQSMGDLGNMLLGLKATMRR 4 P24 GCQSVSEMLRFYTDEVLPRAMQGGGSGGGSKAMGEFDIFINYIEEYLLMR 5 P25 PARLRELR 6 P26 LSSELLDEFKG 7 P27 GEFDIFNYIE 8P28 SLRYYARVRA 9 P29 TNAFSPQ 10 P11 YDDIQKHARRYRVYIRRARDNQTYEVWET 11 P30IQKHARRY 12 P31 NQTYEVWE 13 P32 VASRHIPAM 14 P9* FFKKFFKKFFKKFFKK 15 P6*GTELPSPPSVWFEAEF *P9 and P6 are control peptides (Ni et al.)

Example 4. Basic Plant Expression Constructs for Production of IL-10RAntagonist Peptides in Maize

The amino acid sequences for IL-10R antagonist peptides and all othersequences in this document have been back translated and codon optimizedfor expression in the desired host organism. As an example, for plantexpression, maize codon usage was used to demonstrate codonoptimization, expression cassette assembly, vector assembly, and planttransformation, but other host organisms could also be used. All IL-10Rantagonist peptides and antibodies were back translated and codonoptimized for expression in maize using the computer program Vector NTI(ThermoFisher Scientific, Waltham, Mass.). The resulting DNA sequencesare presented in Table 2 and at the end of the document.

TABLE 2IL-10R antagonist peptides and their maize codon optimized DNA codingsequences Peptide IL-10R antagonist name peptide sequenceMaize codon optimized DNA sequence P21 PARLRELRVKFEEIKDYCCGGCCAGGCTGAGGGAGCTGAGGGTGAAG FQSRDDELNIQLLSSELTTCGAGGAGATCAAGGACTACTTCCAGAGC LDEFKG (SEQ ID NO: 1)AGGGACGACGAGCTGAACATCCAGCTGCTG AGCAGCGAGCTGCTGGACGAGTTCAAGGGC(SEQ ID NO: 16) P22 ENGIYKAMGEFDIFINY GAGAACGGCATCTACAAGGCCATGGGCGAGIEEYLLMRRR (SEQ ID TTCGACATCTTCATCAACTACATCGAGGAGT NO: 2)ACCTGCTGATGAGGAGGAGG (SEQ ID NO: 17) P23 PARLRELRVKFEEIKDYCCGGCCAGGCTGAGGGAGCTGAGGGTGAAG FQGGGSGGGSQQSMGDTTCGAGGAGATCAAGGACTACTTCCAGGGC LGNMLLGLKATMRRGGCGGCAGCGGCGGCGGCAGCCAGCAGAG (SEQ ID NO: 3)CATGGGCGACCTGGGCAACATGCTGCTGGG CCTGAAGGCCACCATGAGGAGG (SEQ ID NO: 18)P24 GCQSVSEMLRFYTDEV GGCTGCCAGAGCGTGAGCGAGATGCTGAGG LPRAMQGGGSGGGSKATTCTACACCGACGAGGTGCTGCCGAGGGCC MGEFDIFINYIEEYLLMATGCAGGGCGGCGGCAGCGGCGGCGGCAG R (SEQ ID NO: 4)CAAGGCCATGGGCGAGTTCGACATCTTCAT CAACTACATCGAGGAGTACCTGCTGATGAGG (SEQ ID NO: 19) P25 PARLRELR CCGGCCAGGCTGAGGGAGCTGAGG (SEQ ID NO: 5)(SEQ ID NO: 20) P26 LSSELLDEFKG CTGAGCAGCGAGCTGCTGGACGAGTTCAAG(SEQ ID NO: 6) GGC (SEQ ID NO: 21) P27 GEFDIFNYIEGGCGAGTTCGACATCTTCAACTACATCGAG (SEQ ID NO: 7) (SEQ ID NO: 22) P28SLRYYARVRA AGCCTGAGGTACTACGCCAGGGTGAGGGCC (SEQ ID NO: 8) (SEQ ID NO: 23)P29 TNAFSPQ ACCAACGCCTTCAGCCCGCAG (SEQ ID NO: 9) (SEQ ID NO: 24) P11YDDIQKHARRYRVYIRR TACGACGACATCCAGAAGCACGCCAGGAGG ARDNQTYEVWETTACAGGGTGTACATCAGGAGGGCCAGGGAC (SEQ ID NO: 10)AACCAGACCTACGAGGTGTGGGAGACC (SEQ ID NO: 25) P30 IQKHARRYATCCAGAAGCACGCCAGGAGGTAC (SEQ ID NO: 11) (SEQ ID NO: 26) P31NQTYEVWE (SEQ ID AACCAGACCTACGAGGTGTGGGAG (SEQ ID NO: 12) NO: 27) P32VASRHIPAM (SEQ ID GTGGCCAGCAGGCACATCCCGGCCATG NO: 13) (SEQ ID NO: 28)

Examples of basic cloning vectors containing individual expressioncassettes for P24 IL-10R antagonist peptide are given in Table 3.Analogous vectors could be made for any of the other IL-10R antagonistpeptides listed in Table 2, or antibodies, by substituting the P24peptide transgene with a different peptide or antibody sequence.

TABLE 3 P24 basic expression vectors Vector Promoter N-terminal signalC-terminal signal pAG4305 prOsGluB4 xGZein27ss KDEL (SEQ ID NO: 29)pAG4981 prZmgZ27 xGZein27ss KDEL (SEQ ID NO: 29) pAG4982 prGtl1xGZein27ss KDEL (SEQ ID NO: 29) pAG4983 prZmGlb1 xGZein27ss KDEL (SEQ IDNO: 29) pAG4984 prZmOle16 xGZein27ss KDEL (SEQ ID NO: 29)

FIGS. 1A-1G illustrate the schematic drawings of the vectors pAG4305(FIG. 1A), pAG4306 (FIG. 1B), pAG4308 (FIG. 1C), pAG4310 (FIG. 1D),pAG4311 (FIG. 1E), pAG4312 (FIG. 1F), and pAG4313 (FIG. 1G).

FIGS. 2A-2D illustrate the schematic drawings of the vectors pAG4981(FIG. 2A), pAG4982 (FIG. 2B), pAG4983 (FIG. 2C), and pAG4984 (FIG. 2D).

Any DNA fragments encoding IL-10R antagonist peptides listed in Table 2,or anti-IL-10 single domain antibodies, can be cloned between desirablepromoter and Nos terminator sequence in the basic P24 peptide expressionvectors (Table 3), in order to generate required expression cassettes.In addition, amino terminal (N) signal sequences, such as xGZein27ss inmaize expression vectors, can be replaced by other signal sequences inorder to modulate specific expression and accumulation of IL-10Rantagonist peptides or antibodies to desired levels. N-terminal signalsequences include, but not limited to, for example by OsGluB4sp (riceGluB-4 glutelin signal peptide), BAASS (barley alpha amylase signalsequence), or PR1 (pathogenesis related protein). The IL-10R antagonistpeptides, or anti-IL-10 single domain antibodies, can be expressed toendoplasmic reticulum (ER) for improved accumulation and glycosylationusing carboxyl terminal (C) retention signal sequences such KDEL (SEQ IDNO: 29), HDEL (SEQ ID NO: 30), or SEKDEL. Furthermore, IL-10R antagonistpeptides, or anti-IL-10 single domain antibodies, can be expressed anddirected to protein storage vacuoles with the help of signal sequencesattached to either N-terminal or C-terminal part of the sequence. Thesestorage vacuole signal sequences include HvAle from barley aleurone(thiol protease) or HvVSD from barley polyamine oxidase. If necessary,IL-10R antagonist peptides, or anti-IL-10 singe domain antibodies, canbe also expressed from basic P24 vectors without signal sequences foraccumulating expressed products in apoplast or cytoplasm. All thosementioned above and other signal sequences of similar functions can beadded to or removed from the basic plant expression vectors. Signalsequences described above can be found in the “List of sequences” at theend of this document.

Example 5. Additional Strategies for Expressing IL-10R AntagonistPeptides in Transgenic Maize

Peptide Concatenation.

This strategy represents expression of a chimeric IL-10R antagonistsequence that contains multiple, contiguously linked, copies of DNAsequences encoding IL-10R antagonist peptides. The peptide codingsequences in a concatemer could be fused directly to one another orseparated by intervening sequences such as AGPA hinges for stabilizingchimeric molecule for expression. Examples of possible variants ofconcatenated peptide sequences for the eight amino acid long peptide P25are provided in Table 4. Each P25 concatemer can be synthesized as DNAmolecule and cloned into any P24 basic expression vectors thuseffectively replacing P24 coding sequence for subsequent expression inmaize. In this way, new maize transformation vectors can be developed,such as for example pAG4306, where P2509 concatemer that is composed ofthree P25 units separated by AGPA hinges is expressed from the riceGluB4 promoter into ER. Nucleotide sequence for P2509 is available inthe “List of sequences” section. A similar approach can be used forexpressing all other short or all IL-10R antagonist peptides that arelisted in Table 2.

TABLE 4 Examples of the P25 IL-10R antagonist peptide andconcatemers for expression in maize PeptideSequence for maize expression P25 PARLRELR (SEQ ID NO: 5) P2501PARLRELRKDEL (SEQ ID NO: 32) P2502 PARLRELRPARLRELR (SEQ ID NO: 33)P2503 PARLRELRPARLRELRKDEL (SEQ ID NO: 34) P2504 PARLRELRAGPAPARLRELR(SEQ ID NO: 35) P2505 PARLRELRAGPAPARLRELRKDEL (SEQ ID NO: 36) P2506PARLRELRPARLRELRPARLRELR (SEQ ID NO: 37) P2507PARLRELRPARLRELRPARLRELRKDEL (SEQ ID NO: 38) P2508PARLRELRAGPAPARLRELRAGPAPARLRELR (SEQ ID NO: 39) P2509PARLRELRAGPAPARLRELRAGPAPARLRELRKDEL (SEQ ID NO: 40)

Gene Fusions.

Another strategy for expressing IL-10R antagonist peptides in plants canemploy chimeric enlargement or gene fusion approach. In this strategy,target peptides can be expressed, for example, as chimeric fusions withparts of the maize gamma-zein 27 kDa. This strategy was used forexpressing zeolin and Zera fusion proteins (Mainieri et al., 2007; U.S.Pat. No. 8,802,825; Llop-Tous, 2010, all of which are incorporatedherein by reference as if fully set forth). Co-expression of targetsequences as gamma-zein fusions allows high level protein accumulationin ER-derived protein bodies. The IL-10R antagonist peptide sequencesselected for expression can be fused to maize gamma-zein sequences withthe help of linker sequences such as, for example linker GSGGSG (SEQ IDNO: 41). Additional linker sequences, for example linkers similar tothose in zeolin fusion protein (GGGGS; SEQ ID NO: 42), Zera fusions(GGGGG; SEQ ID NO: 43), or other linkers can also be exploited (Table5). All IL-10R antagonist peptides can be expressed with or withoutC-terminal sequence such as KDEL (SEQ ID NO: 29), HDEL (SEQ ID NO: 30),SEKDEL (SEQ ID NO: 31), HvVSD, or other such sequences. Furthermore,gamma-zein sequences as fusion components of chimeric proteinenlargements can be substituted for other protein sequences such as, forexample, elastin-like polypeptides (ELP) (Urry, 1992), which were usedfor expressing human IL-10 in tobacco (Patel et al., 2007) orhydrophobins that were used for transient protein expression inNicotiana benthamiana (Joensuu et al., 2010; Jacquet et al., 2014). Wheneither of the latter two approaches is used, expressed protein fusionsform protein bodies. Two constructs that serve as examples forexpressing P2509 concatemer fused with maize gamma-zein components arerepresented by the vectors pAG4308 and pAG4310. The pAG4311 vector is anexample of expressing P2509 as a fusion with 28×VPGVG (SEQ ID NO: 44)elasting-like polypeptide (Conley et al., 2009). Variable number ofrepeats and sequences such as VPGXG in ELP fusion partner could be usedfor expressing IL-10R antagonist peptides. The ELP fusion proteins canbe purified by nonchromatographic bioseparation of recombinant proteins(Lin et al., 2006). The pAG4312 construct provides an example ofexpressing P2509 peptide fused to the mature chain of Trichoderma reeseiHFBI hydrophobin (GenBank Accession #P52754.1). Hydrophobins fusions canbe purified by efficient surfactant-based aqueous two-phase system(ATPS) (Joensuu et al., 2010). Other fusion partners for IL-10Rantagonist peptides could additionally be exploited such as, fusingP2509 to a thermal stable glucanase enzyme, which is presented in vectorpAG4313.

TABLE 5 Linker sequences for developing protein fusions Linker sequenceNucleotide Description GSGGSG ggcagcggcggcagcggc Linker for expression(SEQ ID (SEQ ID NO: 45) Phy02opt: BD21 NO: 41) GGGGS ggcggcggcggcagcLinker used for (SEQ ID (SEQ ID NO: 46) Zeolin expression NO: 42)(Mainieri et al., 2004) GGGGG ggcggcggcggcggc Linker used for ex-(SEQ ID (SEQ ID NO: 47) pressing Zera fusions NO: 43) (Llop-Tous et al.,2011)

Example 6. Production of Single-Domain Antibodies to IL-10

Camelid single-domain antibodies (sdABs; also known as V_(H)Hantibodies) with affinity for cIL-10 were generated by immunizing allama with full-length, purified recombinant cIL-10 (IBI Scientific,Peosta Iowa). Full-length IL-10 was selected, as opposed to individualcIL-10 peptides, because cIL-10 is only 48% identical to the llama IL-10homologue. It was previously unknown whether cIL-10 would generate anadequate immune response in llamas, but given the limited sequenceidentity, cIL-10 was used to test whether llamas would be naïve, andthat the full-length molecule could be used to generate an adequateimmune response. Furthermore, IL-10 is known to dimerize, thus using thefull-length molecule would bias the generated antibodies towardsepitopes that are present in the dimerized molecule. That llamas wouldnot otherwise be exposed to cIL-10, except through injection of isolatedor recombinant cIL-10, provided a novel process for generatinganti-cIL-10 antibodies. Pre-immune serum was collected from a singlellama prior to injection with cIL-10. The first immunization was carriedout with 200 μg of cIL-10 in the presence of Complete Freund's Adjuvant(CFA). Subsequent booster immunizations were carried out, each with 100μg cIL-10, in the presence of Incomplete Freund's Adjuvant (IFA) threeweeks, seven weeks and eleven weeks after the initial immunization.Blood samples (“bleeds”) were collected from the animal one week aftereach of the booster immunizations. FIG. 3 illustrates the llama's immuneresponse prior to (pre-immune), and after being dosed with cIL-10.

The production of antibodies targeting cIL-10 in the animal during thisimmunization process was evaluated via ELISA using each of the bleeds asshown in FIG. 3, and the bleeds were then used to develop single-domainantibodies. The alignment of the chicken and llama IL-10 homologs shows48% identity (68% similarity). The preparation of sdABs has beendescribed elsewhere (Goldman et al. 2006; Arbabi Ghahroudi et al. 1997;Liu et al. 2013). Briefly, peripheral blood mononuclear cells (PBMCs)were isolated from the bleeds that were collected during weeks eight and12 of the immunization protocol. RNA was purified from the PBMCs andused to create phagemid cDNA libraries for expression and screening ofsdABs. Notably, sdABs represent only the heavy chain variable region(VHH) from the llama antibodies produced when the host was injected withrecombinant cIL-10, and therefore the RNA-derived, DNA coding sequenceof sdABs represents a synthetic nucleotide that is produced through anovel process that does not occur in nature. Two rounds of panningagainst purified recombinant cIL-10 were used to enrich the library forphage displaying sdAB with affinity for the antigen. From the enrichedlibraries, individual clones were generated, isolated and sequenced.FIG. 4 illustrates the results of the anti-IL-10 sdAB screening andsequencing. FIG. 4 demonstrates that the isolated sdAB amino acidsequences reside in four distinct groups, with a fifth miscellaneousgroup. The complementarity-determine regions (CDRs) are designated inFIG. 4.

Example 7. Anti-IL-10 sdAbEC50 Measurements and Thermal Stability

Candidate sdABs were evaluated for their apparent binding affinityagainst purified cIL-10, as measured by ELISA. Different concentrationsof individual isolated sdABs were incubated on cIL-10 ELISA plates, withincreasing signals indicative of higher levels of sdAB binding tocIL-10. Apparent EC50 values were estimated by determining the sdABconcentration at which 50% of the maximum signal was observed. FIG. 5illustrates the ELISA measurements and apparent binding affinity ofselected sdAB candidates to chicken IL-10. Among the candidates tested,chIL10sdAB1A11 (SEQ ID NO: 135), chIL10sdAB1F11 (SEQ ID NO: 146), andchIL10sdAB1B9 (SEQ ID NO: 111), had EC50 values of less than 100 nM,with estimated values of 1 nM, 15 nM, and 35 nM, respectively. AnothersdAB, chIL10sdAB1D6 (SEQ ID NO: 143), had an estimated EC50 value of 100nM. Additional sdABs were evaluated in this way for their EC50 values,including chIL10sdAB1H1 (SEQ ID NO: 89) with an EC50 value of 20 nM.Since higher binding affinity is reflected by lower EC50 values, sdABswith low EC50 values, and more divergent sequences were selected forfurther assessment and development.

Although sdABs are considered to have high specificity for theirantigenic target, and do not bind strongly to non-specific peptides, itis often desirable to demonstrate binding specificity to individualepitopes or peptides. The sdABs were prepared using the full-lengthchIL-10 protein, however individual peptides could also be used in ourmethod. Likewise, counter selecting sdABs that bind the full lengthchIL-10, but had little or no affinity to specific peptides was alsoused to identify sdABs that bound desired antigenic peptides, but notothers. Counter selection can be made using several different methods,including an ELISA or dot-blot, where the peptides for counter selectionare immobilized on a surface and the anti-cIL-10 sdAB are incubated toallow binding, washed to remove unbound sdAB, then incubated with alabeled anti-llama antibody to detect any bound sdAB. Any sdAB that doesnot bind the immobilized peptides used for counter selection but stillbind the full length IL-10, can be further developed with the confidencethat they possess the desired binding criteria. Some peptides that wereused for counter selection include: DDELNIQL [peptide 1; SEQ ID NO:180], VLPRAMQT [peptide 2; SEQ ID NO: 181], EKMDENGI [peptide 3; SEQ IDNO: 182], EPTCLHFS [peptide 4; SEQ ID NO: 183], DQMGDLL [peptide 5; SEQID NO: 184], DQLHSLL [peptide 6; SEQ ID NO: 185], VMPKAESD [peptide 7;SEQ ID NO: 186], VMPQAENH [peptide 8; SEQ ID NO: 187], SKLQERGV [peptide9; SEQ ID NO: 188], SELQERGV [peptide 10; SEQ ID NO; 189], ENSCIHFP[peptide 11; SEQ ID NO: 190], DSSCIHLP [peptide 12; SEQ ID NO: 191],DQLNSML [peptide 13; SEQ ID NO: 192], NMLQERGV [peptide 14; SEQ ID NO:193], DSSCTHFP [peptide 15; SEQ ID NO: 194], DDLEIGL [peptide 16; SEQ IDNO: 195], VLPTAIADMTEE peptide 17; SEQ ID NO: 196], TQMEGKGP [peptide18; SEQ ID NO: 197], and NQCCRFV [peptide 19; SEQ ID NO: 198].

Internal screening for thermal stability was performed to determine theheat tolerance of sdABs, which may be important for their use in animalfeed processing. In particular, thermal stability is a highly desirableproperty in animal feed pelleting processes, where the molecules may beexposed to temperatures over 70° C., and up to 125° C., depending on thespecific process and pelleting equipment used. In order to evaluate thethermal stability, heat treated sdABs were prepared by incubating thesdABs at 70° C., 75° C., 80° C., 85° C., and 90° C. for 30 seconds, 60seconds, 90 seconds, 120 seconds, 300 seconds and 600 seconds, and werethen allowed to equilibrate to room temperature. Control sdABs wereincubated at 37° C. or room temperature, for the same period of timethat the heat treated sdABs and also allowed to equilibrate to roomtemperature. The EC50 values of the sdABs were then compared between thecontrol (37° C. or room temperature treated) and treatment (those heatedbetween 70° C. and 90° C. for various amounts of time) sdABs by ELISA.Thermal stability, as expressed by the ratio of the EC50 values of theheat treated sdABs and control sdABs, ranged between 30% to 90%, withhigher thermal stability values correlating to lower temperatures andlower exposure times.

Example 8. Anti-IL-10 sdAb Gastric Stability

Simulated gastric fluid (SGF) consisted of 0.084 M HCl, 35 mM NaCl, pH1.2, containing 2630 Units of pepsin per milliliter. Reaction stopsolution was 200 mM sodium carbonate. Protein samples, including bovineserum albumin (BSA), chIL10sdAB1A11 (SEQ ID NO: 135), chIL10sdAB1F11(SEQ ID NO: 146), chIL10sdAB1H1 (SEQ ID NO: 89), chIL10sdAB1B9 (SEQ IDNO: 111), chIL10sdAB1D6 (SEQ ID NO: 143), chIL10sdAB1E11 (SEQ ID NO:142) chIL10sdAB1F7 (SEQ ID NO: 121) chIL10sdAB1F9 (SEQ ID NO: 141), andchIL10sdAB2A8 (SEQ ID NO: 140), to be tested were brought to aconcentration of 5 mg/mL in storage buffer (50 mM MES, 150 mM NaCl, 40%(v/v) glycerol, pH 6.3).

Single domain antibody samples (2.5 μL) were dispensed into 200 μLthin-walled PCR tubes and prewarmed on a PCR thermal cycler set to 37°C. Aliquots (100 μL) of SGF were placed into PCR tubes and alsoprewarmed. For 0 minute digestion samples, 17.5 μL of stop solution wasadded to the protein samples before adding SGF. Digestions wereinitiated by the addition of 47.5 μL of prewarmed SGF to the sdABsamples. After 1 minute, 2 minutes, 5 minutes, 10 minutes, and 30minutes, reactions were terminated by the addition of 17.5 μL stopsolution. SDS-PAGE sample-loading buffer (17.5 μL; ThermoFisher catalog#NP0007, with dithiothreitol added to a concentration of approximately50 mM) was added to each sample. After heating for 10 minutes at 70° C.,15 μL of each sample was loaded onto a protein electrophoresis gel(ThermoFisher catalog #NP0321 or similar) and electrophoresis wasperformed as directed by the manufacturer. Gels were then stained withCoomassie Blue dye using standard methods. Results are shown in FIG. 6for the 0 and 10 minute time points. In this figure, lane 1—molecularweight standards; lane 2—pepsin only; lane 3—BSA 0 min; lane 4—BSA 10min; lane 5—1A11 0 min; lane 6—1A11 10 min; lane 7—1F11 0 min; lane8—1F11 10 min; lane 9—1H1 0 min; lane 10-1E11 10 min; lane 11-1B9 0 min;lane 12-1B9 10 min; lane 13—molecular weight standards; lane 14-1D6 0min; lane 15-1D6 10 min; lane 16-1E11 0 min; lane 17-1E11 10 min; lane18-1F7 0 min; lane 19-1F7 10 min; lane 20-1F9 0 min; lane 21-1F9 10 min;lane 22-2A8 0 min; lane 23-2A8 10 min; and lane 24—pepsin only. FIG. 6shows that BSA is significantly degraded within a 10 minute digestion inSGF, as are all of the sdABs tested. Given the inherent rapiddigestibility of the sdABs, it is unexpected that the sdABs perform wellin controlling Coccidiosis and binding IL-10 when dosed into feed. Itmay have been anticipated that rapid digestion in SGF would underliepoor performance of sdABs in controlling Coccidiosis as they should bequickly degraded and therefore have a limited ability to bind IL-10 andblock IL-10 signaling. Given that the sdABs described herein areeffective in controlling Coccidiosis suggests that gastric stability isnot a dominating factor in oral antibody administration and is abeneficial trait as proteins that are rapidly degraded in SGF pose alower allergenicity risk than those that are stable in SGF. Given theincreased thermal stability of sdABs (see discussion in Example 1,above), it is unusual that the developed sdABs are readily digestible bypepsin, as it is widely regarded that high thermal stability, asdemonstrated by sdABs, correlates with high SGF stability, in contrastto the measured digestibility of the developed anti-IL-10 sdABs. Theanti-IL-10 sdABs developed herein have good thermal stability and arereadily digested in pepsin, which are attributes that support theproduct's performance and safety profile, which should aid in itsregulatory evaluation and eventual customer acceptance.

Example 9. Chicken IL-10 Ligand-Receptor Assay and sdAB IC50 Measurement

An assay was developed to measure how the sdABs bind their targetcIL-10, and prevent cIL-10 from binding to its receptor. The solubledomain of the cIL-10 receptor was expressed and immobilized on a biacoreprobe surface. It was then incubated with cIL-10 and different mixturesof sdABs and cIL-10, and the binding of cIL-10 to the soluble domain ofthe cIL-10 receptor was measured by surface plasmon resonance.

The amino acid sequence of the soluble domain of the chickeninterleukin-10 receptor subunit 1 (cIL-10R1) was deduced by alignment ofthe amino acid sequence of the human IL-10R alpha receptor subunit(UniProtKB/SwissProt accession number Q13651) with the analogous chickenIL-10 receptor subunit 1 (NCBI reference sequence NP_001034686).

For this assay, the gene coding for the chicken receptor soluble domain(residues 22-231) was synthesized as an upstream fusion to the humanIgG₁ Fc domain (residues 100-330 of UniProtKB/SwissProt accession numberP01857) connected by a linker consisting of IEGRMD [SEQ ID NO: 199] (thefinal, aggregate expressed molecule comprising the cIL-10R1 fused toIEGRMD [SEQ ID NO: 199] fused to the Fc residues 100-330 will bereferred to as “cIL-10R1-Fc”). cIL-10R1-Fc could be directly immobilizedto facilitate the surface plasmon resonance binding assay on a biacoreinstrument. To produce cIL-10R1-Fc, the gene encoding cIL-10R1-Fc wascloned into pGAPZaB (ThermoFisher) via the EcoRI and NotI restrictionsites. Pichia pastoris strain GS115 was transformed with the plasmid asdirected in the pGAPZaB instruction manual. A high-expressing clone wasgrown in a 2.5 L fermenter using a fed-batch protocol. Growth medium inthe batch phase consisted of 1.5 L of 20 g/L peptone, 10 g/L yeastextract, 13.4 g/L yeast nitrogen base, 10 g/L casamino acids, 10 g/Lglycerol, and 100 mM sodium phosphate monobasic. Temperature wasmaintained at 28° C., pH was maintained at 6.0 by addition of 50%ammonium hydroxide, and dissolved oxygen (pO₂) was maintained at 30%.After glycerol was exhausted, as indicated by a spike in dissolvedoxygen, temperature was lowered to 25° C. and feeding of 750 mL of 100g/L glucose, 50 g/L peptone, 25 g/L yeast extract, 10 g/L casaminoacids, 0.5% (v/v) Antifoam 204, and 100 μg/mL zeocin was initiated.

Culture supernatant was isolated by centrifugation and sterile filteredthrough a 0.22 μm filter. Supernatant was taken to 1M ammonium sulfateby the addition of 0.5 volume of 3M ammonium sulfate, 20 mM Tris.HCl, 1mM EDTA, pH 8. After filtering, the receptor fusion was purified byhydrophobic interaction chromatography (Phenyl Sepharose), affinitychromatography (Protein G Sepharose), anion-exchange chromatography(MonoQ), and size-exclusion chromatography.

Chicken IL-10 was obtained from Kingfisher Biotech, Inc. Camelid VHHdomains fused to C-terminal myc- and his-tags were expressed in E. coliwith expression directed to the periplasmic space by an N-terminalsignal peptide. Protein was extracted from the periplasm by osmoticshock and then purified by metal chelation chromatography andsize-exclusion chromatography. Protein concentrations were determined bymeasuring the absorbance at 280 nm, using extinction coefficientscalculated from the amino acid sequences.

Affinity and inhibition were measured using a BIAcore T200 surfaceplasmon resonance instrument (GE Healthcare). Approximately 7000response units of the cIL-10R1-Fc fusion was covalently coupled to a CM5sensor chip using EDC/NHS chemistry as directed by the manufacturer. Allexperiments were conducted at 37° C. in HBS-EP buffer (10 mM HEPES, 150mM NaCl, 3 mM EDTA, 0.005% [v/v] Tween-20, pH 7.4). Binding affinity(K_(d)) for cIL-10 to immobilized cIL-10R1-Fc was determined byinjecting five concentrations of cIL-10 over the sensor chip for 120seconds at 20 μL/second. After 300 seconds of dissociation, the sensorchip was regenerated with a 10 second pulse of 10 mM sodium acetate, 0.5mM EDTA, pH 4 followed by a 120 second stabilization phase before makingthe next injection. The BIAcore evaluation software was used tocalculate the maximum bound material (R_(max)) for each cIL-10concentration. R_(max) values were plotted against cIL-10 concentrationand fit to the equation for a rectangular hyperbola to calculate K_(d).

Inhibition of cIL-10 binding to cIL-10R1-Fc by the sdABs was measured bypreincubating 10 nM cIL-10 with six concentrations of each sdAB inHBS-EP for at least 20 minutes, followed by injection onto the sensorchip as described above. Calculated R_(max) values for each sdABconcentration were plotted against the sdAB concentration to determineIC₅₀ values.

FIG. 7 illustrates anti-IL-10 sdAb IC50 values measured forchIL10sdAB1A11 (SEQ ID NO: 135) and chIL10sdAB1F11 (SEQ ID NO: 146).

Example 10. Cell-Based Assay to Measure the Biological Activity ofcIL-10 on Interferon Gamma (IFN-γ) Production and Inhibition of cIL-10by Anti-IL-10 sdAB in Stimulated Primary Chicken Spleenocytes

IL-10 is known to be a potent regulator of the immune system thataffects many cell types and generally acts to attenuate inflammation andthe immune response (Kevin N. Couper, Daniel G. Blount, Eleanor M.Riley, “IL-10: The Master Regulator of Immunity to Infection,” TheJournal of Immunology, 180:5771-5777, 2008). Primary chicken spleencells were used to evaluate the use of sdABs in blocking the biologicalactivity of IL-10 in cellular signaling on a relevant target cell type.A cell-based assay was developed to study the inhibitory effect ofcIL-10 on the concanavalin A (ConA) dependent induction (orphytohemagglutinin (PHA) dependent induction, both stimulators work withthese cells) of interferon gamma (IFN-γ) production in chicken spleencells (Wu et al. (2016) and Rothwell et al. (2004)). Briefly,lymphocytes and mononuclear cells were isolated from chicken spleens bydifferential centrifugation on Ficoll-Hypaque. Freshly isolated cellswere cultured at 5×10⁶ cells/mL in wells of a 96-well plate for 72 hoursin the presence of 1.2 μg/mL ConA (or 12.5 μg/mL PHA) with, or without,cIL-10 at concentrations of 0-25 mg/mL. Levels of IFN-γ in thesupernatants of treated cells were determined by ELISA. FIG. 8 shows theIFN-γ response of the cells in the absence of ConA, ConA with no cIL-10,and ConA with 0.39, or 1.56, or 6.25, or 25 ng/mL cIL-10). As seen inFIG. 8, IL-10 suppresses of ConA-induced secretion of IFN-γ in primarychicken spleen cells. FIG. 8 also shows that spleen cells have a dosedependent response in IFN-γ production to chIL-10, as increasing cIL-10lowers IFN-γ production in a dose-dependent manner.

To test how effective sdABs were in interrupting cIL-10 stimulatedproduction of IFN-γ, primary chicken spleen cells were incubated withand without different concentrations of sdABs ranging from 0.1 nM up to10 μM, with ConA, and with or without cIL-10. Levels of IFN-γ in theculture supernatants were determined by ELISA. Included in thesestudies, as control treatments, were spleen cells treated only with 5μg/mL ConA (positive control for IFN-γ production), cells treated with 5μg/mL of ConA and 1.5 ng/mL of cIL-10 (positive control for cIL-10inhibition of ConA-dependent IFN-g production), and cells treated with 5μg/mL of ConA, 1.5 ng/mL of cIL-10, and either an anti-IL-10 polyclonalantibody (“aIL10 pAb”, positive control antibody) or a non-specific sdABthat did not bind cIL-10 (“aMOP pAb (NC)”, a negative control antibodyto demonstrate that non-specific binding cannot provide the same effectobserved with antibodies that specifically bind cIL-10). Experimentaltreatments contained 5 μg/mL ConA, 1.5 ng/mL of cIL-10, and varyingconcentrations of anti-IL-10 sdABs. In these experiments, sdABs weredose at 1 nM, 30 nM, and 1000 nM.

FIG. 9 illustrates the anti-IL-10 antibodies (chIL10sdAB1A11 (SEQ ID NO:135), chIL10sdAB1B9 (SEQ ID NO: 111), chIL10sdAB1F11 (SEQ ID NO: 146)effect on the IFN-γ secretion in primary chicken spleen cells. Based onthese results, the apparent EC50 values for chIL10sdAB1A11 (SEQ ID NO:135), chIL10sdAB1B9 (SEQ ID NO: 111), chIL10sdAB1F11 (SEQ ID NO: 146)were measured to be 25 nM, 40 nM, and 60 nM, respectively. Althoughhigher than the EC₅₀ and IC₅₀ values measured for binding of theanti-IL-10 sdABs to cIL-10, they are still in relative agreement withthese values and further demonstrate the biological efficacy of theanti-IL-10 sdABs in blocking IL-10 signaling and decrease immune systemsuppression.

Example 11. Plant Expression of Anti-IL-10 Single Domain Antibodies

Antibody expression was demonstrated in transient expression usingtobacco and in transgenic corn events. Other plant species can be usedto express the anti-IL-10 sdABs, including rice, sorghum, soy beans, andcanola. Depending on the final product and intended use, a particularplant species may be more suited for production than other species.

Expression cassettes containing the sequences of anti-IL-10 sdAbs forexpression in maize are included in Table 6. In Table 6, some vectorscontain single expression cassettes, while other vectors containmultiple expression cassettes, which usually helps increase expressionof the sdAB. The DNA sequence of each chicken anti-IL-10 sdAB containedin the expression cassettes listed in Table 6 has been codon optimizedfor maize gene expression, however, the genes may be optimized for otherplant (or microbial) species to improve their expression when adifferent expression host is desired.

TABLE 6 Plant expression vectors for expression of chicken anti-IL-10sdABs: Vector Chicken anti-IL-10 sdAb expression cassette(s) pAG4314OsGluB4P:xGZein27ss:chIL10sdAB1A11A:KDEL pAG4315OsGluB4P:xGZein27ss:chIL10sdAB1B9:KDEL pAG4316OsGluB4P:xGZein27ss:chIL10sdAB1F11A:KDEL pAG4317OsGluB4P:xGZein27ss:chIL10sdAB1H1A:KDEL pAG4985ZmZ27P:xGZein27ss:chIL10sdAB1A11A:KDEL pAG4986ZmZ27P:xGZein27ss:chIL10sdAB1B9:KDEL pAG4987ZmZ27P:xGZein27ss:chIL10sdAB1F11A:KDEL pAG4988ZmZ27P:xGZein27ss:chIL10sdAb1A11A:KDEL + OsGluB4P:xGZein27ss:chIL10sdAB1A11A:KDEL pAG4989 ZmZ27P:xGZein27ss:chIL10sdAB1B9:KDEL +OsGluB4P:xGZein27ss: chIL10sdAB1B9:KDEL pAG4990ZmZ27P:xGZein27ss:chIL10sdAB1F11A:KDEL + OsGluB4P:xGZein27ss:chIL10sdAB1F11A:KDEL pAG4991 ZmZ27P:xGZein27ss:chIL10sdAB1H1A:KDELpAG4992 ZmZ27P:xGZein27ss:chIL10sdAB1H1A:KDEL + OsGluB4P:xGZein27ss:chIL10sdAB1H1A:KDEL pAG4993 ZmZ27P:xGZein27ss:chIL10sdAB1A11B:KDELpAG4994 ZmZ27P:xGZein27ss:chIL10sdAB1F11B:KDEL pAG4995ZmZ27P:xGZein27ss:chIL10sdAB1H1B:KDEL pAG4996ZmZ27P:xGZein27ss:chIL10sdAB1A11B:KDEL + OsGluB4P:xGZein27ss:chIL10sdAB1A11A:KDEL pAG4997 ZmZ27P:xGZein27ss:chIL10sdAB1F11B:KDEL +OsGluB4P:xGZein27ss: chIL10sdAB1F11A:KDEL pAG4998ZmZ27P:xGZein27ss:chIL10sdAB1H1B:KDEL + OsGluB4P:xGZein27ss:chIL10sdAB1H1A:KDEL

FIG. 10A is a schematic drawing of a vector pAG4314 that includes asingle expression cassette for an anti-IL-10 sdAB(OsGluB4P:xGZein27ss:chIL10sdAB1A11A:KDEL) and the PMI expressioncassette for selection in plants. FIG. 10B is a schematic drawing of avector pAG4988 that increases the transgene dosage by including twoexpression cassettes for the same anti-IL-10 sdAB(ZmZ27P:xGZein27ss:chIL10sdAb1A11A:KDEL+OsGluB4P:xGZein27ss:chIL10sdAB1A11A:KDEL). Vector pAG4988 also includes the PMIexpression cassette for selection in plant tissues. The T-DNA sequencesfor the vectors listed in the table are provided below in such a waythat each sequence starts with the right border repeat and ends with theleft border repeat.

Deduced protein sequences for selected chicken IL10 sdAb are providedwith the maize gamma zein 27 signal sequence and KDEL signal sequence,which are underlined at N-terminal and C-terminal ends of the protein,respectively.

Protein Sequences of chIL10 sdABs Encoded by Plant Expression Cassettes

>xGZein27ss: chIL10sdAb1A11: KDEL (SEQ ID NO: 84)MRVLLVALALLALAASATSQVQLQESGGGLVQPGGSLRLSCASGNIFSINTMGWYRQAPGKQRELVASITTGGTTNYEDSVKGRFTISRDNAKKTVYLQMNRLKPEDTAVYYCNHRRSYSGRDYPVYGMDYWGKGTLVTVSSKDEL >xGZein27ss: chIL10sdAb1B9: KDEL(SEQ ID NO: 85) MRVLLVALALLALAASATSQVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAWGWFRQAPGKEREFVARISFSGGHTYYSDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADPTPYGLRNERNYPYWGQGTQVTVSSKDEL >xGZein27ss: chIL10sdAb1F11: KDEL(SEQ ID NO: 86) MRVLLVALALLALAASATSQVQLQEFGGGLVQPGGSLRLSCASGRTGSSYAMGWFRQAPGKEREFVAAISWSGGSTDYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAVDRNLFKLRVAVQEYTNLGQGTQVTVSSKDEL >xGZein27ss: chIL10sdAb1H1: KDEL(SEQ ID NO: 179) MRVLLVALALLALAASATSQVQLQASGGGLVQAGGSLRLSCAASGRTFNSYAWGWFRQAPGKERGFVARISFSGGHTYYSDSVKGRFTISRDNAKNSVYLQMNSLKPEDTAVYYCAADPTPYGLRNERNYHYWGQGTQVTVSSKDEL

The nucleotide sequences encoding chIL10sdAB1A11, chIL10sdAB1F11, andchIL10sdAB1H1 antibodies in vectors and nucleotide sequences were named1A11A (chIL101A11A), 1F11A (chIL101F11A), and 1H1A (chIL101H1A),respectively, to reflect different coding sequences with altered codonusage. This modification was made in order to avoid any possibleconfusion in the future due to availability of different variants (forexample, “variant A” and “variant B”) of the maize codon optimizedsequences for maize expression. The deduced protein sequences encoded bythe variants “A” and “B” are identical.

Nucleotide sequence alignments of the maize codon optimized variants “A′and “B” of the selected chIL10 sdABs:

CLUSTAL O(1.2.4) multiple sequence alignments 1A11ACAGGTTCAGCTGCAGGAAAGCGGTGGCGGACTGGTGCAGCCAGGTGGCAGCCTCAGGCTG 60 1A11BCAGGTGCAGCTCCAGGAGTCCGGCGGCGGCCTCGTGCAGCCGGGCGGCTCCCTCCGCCTG 60***** ***** *****   *** ***** ** ******** ** ***  **** * *** 1A11AAGCTGCGCTGCTAGCGGCAATATTTTTAGCATTAACACAATGGGTTGGTATAGACAGGCT 120 1A11BAGCTGCGCCGCGTCCGGCAACATCTTCAGCATCAACACGATGGGCTGGTACAGGCAGGCC 120******** **   ****** ** ** ***** ***** ***** ***** ** ***** 1A11ACCTGGCAAGCAGCGTGAGCTCGTTGCCAGCATTACCACGGGTGGTACAACCAATTATGAA 180 1A11BCCCGGCAAGCAGCGGGAGCTCGTGGCCTCCATCACCACGGGCGGCACCACGAACTACGAG 180** *********** ******** ***  *** ******** ** ** ** ** ** ** 1A11AGATAGCGTGAAGGGTCGTTTTACCATTAGCAGGGACAATGCTAAGAAGACCGTTTACCTC 240 1A11BGACAGCGTCAAGGGCCGCTTCACCATCTCCAGGGACAACGCCAAGAAGACGGTGTACCTC 240** ***** ***** ** ** *****   ********* ** ******** ** ****** 1A11ACAGATGAACAGGCTGAAGCCAGAAGATACCGCCGTGTATTACTGCAACCACAGGAGAAGC 300 1A11BCAGATGAACCGCCTGAAGCCGGAGGACACGGCGGTCTACTACTGCAACCACCGCAGGTCC 300********* * ******** ** ** ** ** ** ** ************ * **   * 1A11ATATAGCGGAAGAGATTATCCTGTTTACGGTATGGACTACTGGGGCAAGGGAACCCTGGTT 360 1A11BTACAGCGGCAGGGACTACCCCGTGTACGGCATGGACTACTGGGGCAAGGGCACCCTCGTG 360** ***** ** ** ** ** ** ***** ******************** ***** ** 1A11AACCGTGAGCAGC 372 [SEQ ID NO: 173] 1A11BACCGTGTCCTCC 372 [SEQ ID NO: 174] ******  *  * 1F11ACAGGTTCAGCTCCAGGAGTTTGGTGGCGGACTGGTGCAGCCAGGTGGCAGCCTCAGGCTG 60 1F11BCAGGTGCAGCTCCAGGAGTTCGGCGGCGGCCTCGTGCAGCCGGGCGGCTCCCTCCGCCTG 60***** ************** ** ***** ** ******** ** ***  **** * *** 1F11AAGCTGCGCTGCTAGCGGTAGAACCGGCAGCAGCTATGCTATGGGATGGTTTAGACAGGCT 120 1F11BAGCTGCGCCGCGTCCGGCAGGACGGGCTCCAGCTACGCGATGGGCTGGTTCAGGCAGGCG 120******** **   *** ** ** ***  ****** ** ***** ***** ** ***** 1F11A.CCAGGCAAGGAGCGTGAATTTGTTGCTGCCATTAGCTGGAGCGGAGGTAGCACCGATTAT 180 1F11BCCCGGCAAGGAGAGGGAGTTCGTGGCGGCCATCTCGTGGAGCGGCGGCAGCACCGACTAC 180** ********* * ** ** ** ** *****    ******** ** ******** ** 1F11AGCTGACAGCGTGAAGGGCAGGTTTACCATTAGCAGAGATAATGCCAAGAACACCATGTAC 240 1F11BGCTGACTCCGTCAAGGGCCGCTTCACCATCAGCAGGGACAACGCGAAGAACACGATGTAC 240******  *** ****** * ** ***** ***** ** ** ** ******** ****** 1F11ACTCCAGATGAATAGCCTGAAGCCAGAGGATACCGCTGTTTATTACTGCGCCGTGGACCGT 300 1F11BCTCCAGATGAACTCCCTGAAGCCGGAGGACACCGCCGTGTACTACTGCGCGGTCGACCGC 300***********   ********* ***** ***** ** ** ******** ** ***** 1F11AAATCTCTTTAAGCTGAGGGTTGCTGTGCAGGAATACACCAACCTCGGCCAGGGAACCCAG 360 1F11BAACCTCTTCAAGCTGAGGGTGGCCGTCCAGGAGTACACCAACCTCGGCCAGGGCACCCAG 360** ***** *********** ** ** ***** ******************** ****** 1F11AGTTACCGTGAGCAGC 373 [SEQ ID NO: 173] 1F11BGTGACCGTGTCCTCC 373 [SEQ ID NO: 176] ** ******  *  * 1H1ACAGGTTCAGCTCCAGGCTTCGGGCGGCGGGCTCGTCCAGGCGGGCGGCTCGCTCAGGCTC 60 1H1BCAGGTGCAGCTCCAGGCCTCCGGCGGCGGCCTCGTGCAGGCGGGCGGCTCCCTCCGCCTG 60***** *********** ** ******** ***** ************** *** * ** 1H1ATCGTGCGCGGCGTCGGGGCGGACTTTCAACAGCTACGCTTGGGGCTGGTTCAGGCAGGCG 120 1H1BAGCTGCGCCGCGTCCGGCAGGACCTTCAACAGCTACGCTTGGGGCTGGTTCAGGCAGGCG 120   ***** ***** **  **** ************************************ 1H1ACCGGGCAAGGAGCGCGGCTTCGTGGCCAGGATCTCCTTCAGCGGCGGCCACACCTACTAC 180 1H1BCCGGGCAAGGAGCGCGGCTTCGTGGCCAGGATCTCCTTCAGCGGCGGCCACACCTACTAC 180************************************************************ 1H1ATCCGACAGCGTCAAGGGCCGCTTCACGATCTCCAGGGACAACGCCAAGAACAGCGTGTAC 240 1H1BTCCGACAGCGTCAAGGGCCGCTTCACGATCAGCAGGGACAACGCCAAGAACTCCGTGTAC 240******************************  *******************  ******* 1H1ACTCCAGATGAACTCCCTGAAGCCCGAGGACACGGCCGTCTACTACTGCGCGGCGGACCCG 300 1H1BCTCCAGATGAACAGCCTGAAGCCCGAGGACACGGCCGTCTACTACTGCGCGGCGGACCCG 300************  ********************************************** 1H1AACGCCCTACGGCCTCAGGAACGAGCGGAACTACCATTACTGGGGGCAGGGCACGCAGGTC 360 1H1BACCCCATACGGCCTCCGCAACGAGAGGAACTACCACTACTGGGGCCAGGGCACCCAGGTG 360** ** ********* * ****** ********** ******** ******** ***** 1H1AACTGTCTCTTCG 372 [SEQ ID NO: 177] 1H1B ACCGTGTCCTCC 372 [SEQ ID NO: 178]** ** ** **

TABLE 7 The percentage of nucleotide sequence identity between “A” and“B” variants Sequence Sequence Sequence “A” “B” identity, % 1A11A 1A11B78.2 1F11A 1F11B 79.7 1H1A 1H1B 90.3

Any polynucleotides encoding anti-IL10 antibodies can be cloned betweendesirable promoter and terminator sequences in the plant expressionvectors described herein (Table 6), in order to generate expressioncassettes. In addition, amino terminal (N) signal sequences, such asxGZein27ss in maize expression vectors, can be replaced by other signalsequences in order to modulate specific expression and accumulation ofanti-IL-10 antibodies to desired levels. N-terminal signal sequencesinclude, but not limited to, for example by OsGluB4sp (rice GluB-4glutelin signal peptide), BAASS (barley alpha amylase signal sequence),or PR1 (pathogenesis related protein). The anti-IL-10 antibodies can beexpressed to endoplasmic reticulum (ER) for improved accumulation andpotential glycosylation using carboxyl terminal (C) retention signalsequences such KDEL (SEQ ID NO: 29), HDEL (SEQ ID NO: 30), or SEKDEL(SEQ ID NO: 31). Furthermore, anti-IL10 antibodies can be expressed anddirected to protein storage vacuoles with the help of signal sequencesattached to either N-terminal or C-terminal part of the sequence. Thesestorage vacuole signal sequences include HvAle from barley aleurone(thiol protease) or HvVSD from barley polyamine oxidase. If necessary,anti-IL-10 antibodies can be also expressed from expression vectorswithout signal sequences for accumulating expressed products inapoplast, chloroplast, or cytoplasm. All of the genetic elementsmentioned above, including other signal sequences with similarfunctions, can be added to or removed from the basic plant expressionvectors to tailor the expression properties of the anti-IL-10 sdAB. FIG.11 illustrates that using the genetic elements described herein, sdABscan be expressed at high level in corn grain. In FIG. 11, sdABsexpressed in corn were extracted from individual seed from a hemizygousparent, where 50% of the seed contained the expression cassette and 50%of the seed did not contain the expression cassette. In FIG. 11,individual seed from two different transgenic events were genotyped andanalyzed for the presence of the expressed sdAB by SDS-PAGEelectrophoresis and coomassie staining. As shown in FIG. 11, thepresence of the sdAB correlated perfectly with the genotyping result,that is, only seed that tested positive for the gene by PCR produced aprotein band at the right size of the sdAB. Further, expression levelsof the sdAB in event 1 were estimated at 3 mg per gram of corn grain,which would result in an expression level of 9 mg of sdAB per gram ofcorn for the fully homozygous event. Expression levels for recombinantproteins, including sdABs, up to 21 mg per gram of corn grain have beenobserved using the expression cassettes and genetic elements describedherein.

Example 12. Transient Expression of Chicken IL10 sdAb 1A11 in Leaves ofTobacco Nicotiana benthamiana

Transient protein expression in plants has been used by multiple groupsfor production of therapeutic proteins and vaccine antigens. Amongvarious plant species, tobacco Nicotiana benthamiana, is one of the mostsuitable production hosts because it can achieve a high level of proteinexpression in a short timeframe by using a leaf infiltration procedure.Such production attributes are required for economical heterologousprotein production.

Genetic Elements and Construction of Vectors

For expression in N. benthamiana, the chicken IL-10 sdAb 1A11 (referredto herein as Nb1A11; SEQ ID NO: 202) sequence was codon optimized forNicotiana codon usage and synthesized by GenScript as either an 868 bpNcoI-AvrII DNA fragment, which at 5′ end contained 90 bp tobacco PR1agene sequence [SEQ ID NO: 205] encoding transit peptide, 304 bp firstintron of Arabidopsis ubiquitin 10 gene (AtUBQ10i) in Nb1a11 codingregion, and at 3′ end myc tag, 6×His, and KDEL sequences [SEQ ID NO:206], or as 564 bp NcoI-AvrII fragment without the AtUBQ10i intron. TheNb1A11:AtUBQ10i sequence is shown below as SEQ ID NO: 203, and theintron sequence is indicated by the bold characters and is underlined.

[SEQ ID NO: 203]CAAGTTCAGTTACAGGAAAGCGGGGGAGGTTTAGTTCAGCCTGGGGGTTCATTGAGGTTGAGTTGTGCAGCAAGTGGAAATATTTTTTCTATTAATACTATGGGATGGTATAGACAAGCTCCAG

GAAAGCAAAGAGAACTTGTTGCAAGTATTACTACTGGAGGAACTACAAATTACGAAGATAGTGTTAAAGGAAGATTCACTATTTCAAGAGATAATGCTAAGAAAACAGTTTATCTTCAGATGAATAGATTGAAGCCAGAAGATACAGCAGTTTACTACTGTAATCATAGAAGATCATACTCTGGTAGAGATTATCCTGTTTATGGTATGGATTATTGGGGAAAAGGGACATTAGTTACAGTTAGCAGC

The AtUBQ10i was inserted into Nb1A11 coding region between nucleotides124 and 125 for dual purpose: 1) monitoring expression of Nb1A11 fromplant cells rather than from Agrobacterium; 2) potentially enhancingexpression of Nb1A11 in tobacco, since positive effects of heterologousintrons on gene transcription in plants and other species are welldocumented in the literature (“Introns increase gene expression incultured maize cells,” J. Callis, M. Fromm, V. Walbot, Genes Dev.,1:1183-1200, 1987; doi:10.1101/gad.1.10.1183; “Increased Gene Expressionby the First Intron of Maize Shrunken-1 Locus in Grass Species,” V.Vasil, M. Clancy, R. J. Ferl, I. K. Vasil, L. C. Hannah, Plant Physiol.91:1575-1579, 1989; “Intron-mediated enhancement as a method forincreasing transgene expression levels in barley,” J. G. Bartlett, J. W.Snape, W. A. Harwood, Plant Biotechnology Journal, 7:856-866, 2009, allof which are incorporated herein by reference as if fully set forth).The sequences of myc tag and 6×His were included to facilitate Nb1A11sdAB detection and purification. The KDEL sequence was added forretaining the expressed 1A11 sdAb in endoplasmic reticulum (ER) forimproving accumulation levels of the protein. For transient expressionof Nb1A11 sdAB in N. benthamiana, a new and previously uncharacterizedconstitutive ubiquitin 1 gene promoter (prNbUbi1) was used. The sequenceof the prNbUbi1 is shown below as SEQ ID NO: 204, wherein the sequenceof the intron sequence is indicated by the bold characters and isunderlined.

[SEQ ID NO: 204]CATGAAAGTCCACATCATCAGCTCGTCCCAAACATCACTACTAGACCCAACTCGTTCAATCTTCTCGACTACAACAAATGAAATCCGCTCATCAAGGTGTCTGAGGCTGATCTCAATAAATGGAGGGACTAATTGTATGGATCGAAATCTGCCCCAAAATATTTAGGGTAAGGTACATTGAAGAAAGAGTCATCGAGGTCGATCAGGAAACGATCGAGATGTTAACAATGGTCGATGTCGAGCACCGCATGTAGAGTTGTAACACCTAGTTTTTAGAATAGGATAATACAAAGAATATTCTATTGGATATCCTTTACACTTATATTATTAGAGTTTGTTAGGAAAATGACCCACATAAATAGGAAAAAAGACAATGAATGGAGACAGGTGACATTTATCTGATGAGAACAGACTTTTGATAGAAGATATTTTCTCTCTCACTAAGATACAAACACTACATTTTCATCAAGATTCTTGTTCATATCATTGTACACTTTTCTATCAAATCTGAAATAATTTAAATATTCTAGGATTTGTCTGTCACTCATCATTGTCAGACGGGATAATCATGTACTCATCCTTTTTTGGCAAACCACTTTTTCTATTTACTTAAATGCCATTTATTGATATCTATTGCTAGTCATTCCTCCACCGTTGCTCATACTTTTTTGCAATAGTATGCATGTTGATATCAATCCACCACCAAATCTTCTAACATTAATCATATTTTCACAACTTACATTTATAAATATTATTATTAACTAAGTTTAACTCACTATTATATAAACTCAATTGTTTTACTCGAAAGTTACACTATTATATTGAGAATTACGTTTCCAAACTTTTTAAGCATTTATTGTGTAACCATAAGAGACTTTGATTTTTTAAAAATTATTTAGATTTTATTAATGAGAATGGCACAACATTATGGTCAACTATGTATTTCATCATTAACTAAATAGTTAGCACTTTGATTCTTTCACATGAATTATGAATTTATGATGGGCTCAAATTAAAATTAAATTATTCACAAAAACTTATTTTTATATTCTACGACACCCACTTTTCTAGCTTTTTCCCGAAGGGGCGTGAGAGTGTCACACACGCTCCAAATTTCCCAACCAAACAAGGAAAGGGCAGAGAAAGATAGCTTTAGCGTGTTGTTTTGGTGCACTACACGTCATTAGGACACGTGTCATGATATAATAGGCCAATCCCACGAGGCGGTTTCGTCTTGAGTCGGCCATAGTGTCCATAAATGAGGGCTCTCCGTCGGTTTCCCCATCATTCATCAGATTTATCTTCTATACTTCATCGCCTTCATATTTCTCTCTCAAG

This promoter was identified by screening N. benthamiana ExpressedSequence Tag (EST) database for the most abundant in leaf tissueubiquitin gene transcript. The database is maintained by the Nicotianabenthamiana Genome and Transcriptome Sequencing Consortium (Nakasugi K,Crowhurst R N, Bally J, Wood C C, Hellens R P, Waterhouse P M (2013) DeNovo Transcriptome Sequence Assembly and Analysis of RNA Silencing Genesof Nicotiana benthamiana. PLoS ONE 8(3): e59534, which is incorporatedby reference herein as if fully set forth). The transcriptNbv6.1trP26199, annotated as putative ubiquitin 1, appeared to containsignificantly larger number of ESTs (1196) than other ubiquitin relatedtranscripts. The Nbv6.1trP26199 specific 1466 bp upstream genomicsequence, which included 128 bp 3′UTR positioned intron, was identifiedin N. benthamiana draft genome sequence (v1.0.1) that is available atthe Sol Genomics Network at Boyce Thompson Institute for Plant Research(Bombarely, A., H. G. Bosh, J. Vrebalov, P. Moffett, L. A. Mueller, andG. B. Martin (2012), A draft genome sequence of Nicotiana benthamiana toenhance molecular plant-microbe biology research. MolecularPlant-Microbe Interactions 25:1523-1530, which is incorporated byreference herein as if fully set forth).

This 1466 bp sequence in N. benthamiana genome has nucleotidecoordinates 74703-76169 in the Scaffold No: 5041 and is fused to codingregion of a putative ubiquitin gene that encodes five 76 amino acid longidentical ubiquitin monomers. The 1466 bp prNbUbi1 was synthesized byGenScript as NotI-NcoI fragment. The entire Nb1A11+AtUBQ10i or Nb1A11expression cassettes were cloned into NotI-KpnI sites of the pLH9000vector, which was kindly provided by Dr. I. Lernomtova (IPK Gatersleben,Germany), and the final constructs were designated as pLH1A11int orpLH1A11 respectively. FIG. 12 illustrates pLH1A11int expressioncassette. FIG. 13 illustrates pLH1A11 expression cassette. Subsequently,pLH9000, pLH1A11int and pLH1A11 were electroporated intoelectrocompetent cells of the Agrobacterium strain GV3101. Agrobacteriumcolonies carrying pLH9000, pLH1A11int or pLH1A11 constructs werevalidated by PCR.

N. benthamiana Plant Growing and Inoculation with Agrobacterium

The seeds of N. benthamiana were acquired from The US NicotianaGermplasm Collection (NC State University). The seeds were sowed into4″×4′ pots containing ProMix soil. After germination the seedlings andthe plants were kept at 16 h day and 8 h night light regime. Five weeksold healthy N. benthamiana plants were used for syringe infiltrationwith Agrobacterium strains GV3101 harboring either pLH9000 as a negativecontrol, pLH1A11int or pLH1A11 expressing Nb1A11 (chicken IL-10 sdAb1A11for N. benthamiana expression as described above), or with the mixtureof two Agrobacterium strains, such as GV3101 with pLH1A11int and C58C1with p19. As used herein, 1A11 sdAB is synonomous with anti-chickenIL-10 sdAB and chicken IL-10 sdAb1A11. The p19 is a tomato bushy stuntvirus protein, which is involved into suppression of RNA-dependent genesilencing thus improving expression of heterologous proteins.Agrobacterium strains GV3101 and C58C1 with p19 were kindly provided byDr. I. Lernomtova (IPK Gatersleben, Germany). The Agrobacterium strainscontaining plasmids were grown from single colonies overnight in LBmedium supplemented with corresponding antibiotics, the cells wereharvested by centrifugation and resuspended to OD₆₀₀=0.4 in 10 mM MgCl₂,10 mM MES-K (pH 5.6). Prior to syringe infiltration of N. benthamianaleaves 100 μM Acetosyringone was added to each Agrobacterium strain. Theleaf tissues for expression analysis of Nb1A11 were harvested on day 4post infiltration.

RNA Analysis of Nb1A11 Expression

The total plant RNA from Agrobacterium infiltrated N. benthamiana leaftissues was isolated with NucleoSpin RNA Plant Kit (Takara) according tomanufacturer's protocol. Subsequently, 1 μg of the total RNA wasconverted into cDNA using iScript cDNA Synthesis Kit (Bio-Rad) and 1.5μl of each cDNA was used as template in PCR reactions with the followingprimers:

forward primer 3661 (5′-CGTGCCCAAGTTCAGTTACA-3′ [SEQ ID NO: 200]), and

reverse primer 3662 (5′-TTGCAACAAGTTCTCTTTGCTT-3′ [SEQ ID NO: 201]). Theprimers were positioned to flank intron AtUBQ10i within the codingregion of Nb1A11 and allow unambiguous identification of the plant cellexpressed 1A11 transcript with the fully spliced out intron. ThePlatinum Taq DNA Polymerase (Invitrogen) was used to amplify PCRproducts under conditions recommended by manufacturer with 36 cycles ofamplification and primer annealing temperature of 55° C. The PCRproducts were resolved on 2% agarose gel. FIG. 14 illustrates end pointRT-PCR analysis of transiently expressed Nb1A11 in N. benthamianaleaves. In this figure, lanes 1-5: lane 1—GV3101+pLH9000 (negativecontrol); lane 2—GV3101+pLH1A11int; lane 3—GV3101+pLH1A11; lane4—plasmid pLH1A11int; and lane 5—plasmid pLH1A11.

In N. benthamiana leaf tissues infiltrated with the negative controlplasmid pLH9000 no 1A11 transcripts were amplified (lane 1). DistinctPCR products of the expected 1A11 transcript sizes were amplified fromN. benthamiana leaf tissues infiltrated with either pLH1A11int orpLH1A11 (lanes 2 and 3 respectively).

Amplified PCR products from plasmids pLH1A11int and pLH1A11 were uses aspositive controls and run in lanes 4 and 5. The resulting products inlanes 4 and 5 were observed to have identical sizes to the products inlanes 2 and 3. A lower intensity PCR band corresponding in size to theexpected 458 bp fragment containing AtUBQ10i was also detected in lane2. This fragment could have been amplified from either N. benthamianagenomic DNA, which was still lingering in total RNA preparation of thesample despite of its removal by DNase digestion as suggested bymanufacturer's instructions, or alternatively, the amplification productis indicative of a fraction of isolated total RNA containing Nb1A11transcripts with still unspliced AtUBQ10i.

1A11 sdAb Transient Protein Expression Analysis

Based on the results of Nb1A11 RNA transcript analysis in leaves of N.benthamiana, the leaf tissue samples that were infiltrated withpLH1A11int and pLH1A11int+p19 were selected for protein isolation andWestern blot analysis. Agrobacterium-infiltrated leaf tissues wereground in liquid nitrogen and the total protein was isolated usingextraction buffer composed of 1 M NaCl, 50 mM Sodium Phosphate p118.0,10 mM Imidazole. The extraction buffer was supplemented with 1× HaltProtease Inhibitor Cocktail (ThermoFisher) and 2 mM β-mercaptoethanol.Protein extraction was performed at 4° C. for 1 h with agitation,samples were centrifuged twice to remove plant debris. The 1A11 sdABcontaining 6×His tag at its C-terminal end was isolated from the clearedsupernatant of the total N. benthamiana leaf protein using Ni-NTA spincolumns (QIAGEN) according to manufacturer's protocol for nativeconditions. The 1A11 sdAB was further concentrated using Amicon Ultra-2centrifugal filters (Millipore-Sigma) and protein concentration wasdetermined by NanoDrop spectrophotometer. Subsequently, 15 μg of 1A11containing concentrated protein fraction was resolved on 4-12% gradientNuPAGE polyacrylamide gels (ThermoFisher) using 1×MOPS SDS gel runningbuffer. Biotynylated Protein Ladder (Cell Signalling Technologies) andPrecision Plus Protein Kaleidoscope (Bio-Rad) were used as molecularweight standards. The proteins were separated in a polyacrylamide geland transferred onto PVDF membrane using semi-dry Western blottingprocedure. The PVDF membrane bound 1A11 protein was detected usingRabbit Anti-VHH HRP (Invitrogen) as the primary antibody at 1:2500dilution followed by Anti-Rabbit IgG Peroxidase Goat (Sigma) as thesecondary antibody at 1:5000 dilution. Detection of the biotinylatedproteins in the protein molecular weight ladder was accomplished byAnti-biotin HRP-liked Ab at 1:15000 dilution (Cell SignalingTechnology). The signal detection was achieved using Super Signal WestPico Plus chemiluminescent substrate (ThermoFisher). FIG. 15 illustrates1A11 protein expression in Agrobacterium infiltrated leaves of N.benthamiana. The Western blot shows detection of 1A11 sdAb in samples 5,6. It was demonstrated that the lanes 5 and 6 contain a protein ofexpected molecular weight of 17.4 kDa that is cross reactive with theAnti-VHH primary antibody, indicating the protein is indeed 1A11 sdAB.

Example 13. Anticoccidial Efficacy of IL-10R Peptide Antagonists andsdABs in Commercial Broiler Chickens Infected with a Mixture of Eimeriaacervulina, E. maxima, and E. tenella Field Isolates

The same study design was used to measure the anticoccidialefficacy/sensitivity anti-IL10 antibodies, or IL-10R antagonists againsta mixture of Eimeria acervulina, E. maxima, and E. tenella. In thesetrials, chickens were separated into multiple control groups that wereeither exposed to Eimeria (Infected, I) or not exposed to Eimeria(Non-Infected, NI), and treatment groups that were exposed to Eimeriaand treated with various diets. The control groups included a negativecontrol group receiving normal feed with and without anti-IL-10 sdAB (orwith and without IL-10R antagonist) in the feed, and a positive controlgroup receiving a standard chemical Coccidiostat. Treatment groups werefed diets containing from 50 g to four kilograms of milled grainexpressing anti-IL-10 sdAB per kilogram of feed (or IL-10R antagonistpeptide doses ranging from 1 milligram of peptide per kilogram of feedto 40 milligrams of IL-10R antagonist peptide per kilogram of feed.Anti-IL-10 sdABs, including SEQ ID NO: 87 (chIL10sdAB1115), SEQ ID NO:88 (chIL10sdAB1E9), SEQ ID NO: 89 (chIL10sdAB1H1), SEQ ID NO: 90(chIL10sdAB1G6), SEQ ID NO: 91 (chIL10sdAB1C10), SEQ ID NO: 92(chIL10sdAB1B6), SEQ ID NO: 93 (chIL10sdAB1D12), SEQ ID NO: 94(chIL10sdAB1C2), SEQ ID NO: 95 (chIL10sdAB1B5), SEQ ID NO: 96(chIL10sdAB1E2), SEQ ID NO: 97 (chIL10sdAB1G7), SEQ ID NO: 98(chIL10sdAB1G9), SEQ ID NO: 99 (chIL10sdAB1H12), SEQ ID NO: 100(chIL10sdAB2A9), SEQ ID NO: 101 (chIL10sdAB1E12), SEQ ID NO: 102(chIL10sdAB1E10), SEQ ID NO: 103 (chIL10sdAB1F12), SEQ ID NO: 104(chIL10sdAB1A8), SEQ ID NO: 105 (chIL10sdAB1C8), SEQ ID NO: 106(chIL10sdAB1C12), SEQ ID NO: 107 (chIL10sdAB1B1), SEQ ID NO: 108(chIL10sdAB1F1), SEQ ID NO: 109 (chIL10sdAB1D11), SEQ ID NO: 110(chIL10sdAB1E6), SEQ ID NO: 111 (chIL10sdAB1B9), SEQ ID NO: 112(chIL10sdAB1B10), SEQ ID NO: 113 (chIL10sdAB1F5), SEQ ID NO: 114(chIL10sdAB1A6), SEQ ID NO: 115 (chIL10sdAB1D5), SEQ ID NO: 116(chIL10sdAB1D8), SEQ ID NO: 117 (chIL10sdAB1B4), SEQ ID NO: 118(chIL10sdAB1C7), SEQ ID NO: 119 (chIL10sdAB1B3), SEQ ID NO: 120(chIL10sdAB1D7), SEQ ID NO: 121 (chIL10sdAB1F7), SEQ ID NO: 122(chIL10sdAB1F10), SEQ ID NO: 123 (chIL10sdAB1F2), SEQ ID NO: 124(chIL10sdAB1F3), SEQ ID NO:125 (chIL10sdAB1F8), SEQ ID NO: 126(chIL10sdAB1C9), SEQ ID NO: 127 (chIL10sdAB1A12), SEQ ID NO: 128(chIL10sdAB1C3), SEQ ID NO: 129 (chIL10sdAB1E7), SEQ ID NO: 130(chIL10sdAB1D9), SEQ ID NO: 131 (chIL10sdAB1A9), SEQ ID NO: 132(chIL10sdAB1H10), SEQ ID NO: 133 (chIL10sdAB1C1), SEQ ID NO: 134(chIL10sdAB1D1), SEQ ID NO: 135 (chIL10sdAB1A11), SEQ ID NO: 136(chIL10sdAB1G8), SEQ ID NO: 137 (chIL10sdAB1A5), SEQ ID NO: 138(chIL10sdAB1C5), SEQ ID NO: 139 (chIL10sdAB1H6), SEQ ID NO: 140(chIL10sdAB2A8), SEQ ID NO: 141 (chIL10sdAB1F9), SEQ ID NO: 142(chIL10sdAB1E11), SEQ ID NO: 143 (chIL10sdAB1D6), SEQ ID NO: 144(chIL10sdAB1C4), SEQ ID NO: 145 (chIL10sdAB1H4), SEQ ID NO: 146(chIL10sdAB1F11), SEQ ID NO: 147 (chIL10sdAB1D3), SEQ ID NO: 148(chIL10sdAB1A7), SEQ ID NO: 149 (chIL10sdAB1H8), SEQ ID NO: 150(chIL10sdAB1H3), SEQ ID NO: 151 (chIL10sdAB1B8), SEQ ID NO: 152(chIL10sdAB1B2), SEQ ID NO: 153 (chIL10sdAB1D2), or SEQ ID NO: 154(chIL10sdAB1D10), or peptides of SEQ ID NO: 1 [P21], SEQ ID NO: 2 [P22],SEQ ID NO: 3 [P23], SEQ ID NO: 4 [P24], SEQ ID NO: 5 [P25], SEQ ID NO: 6[P26], SEQ ID NO: 7 [P27], SEQ ID NO: 8 [P28], SEQ ID NO: 9 [P29], SEQID NO: 10 [P11], SEQ ID NO: 11 [P30], SEQ ID NO: 12 [P31], SEQ ID NO: 13[P32] SEQ or concatenated peptides SEQ ID NO: 32 [P2501], SEQ ID NO: 33[P2502], SEQ ID NO: 34 [P2503], SEQ ID NO: 35 [P2504], SEQ ID NO: 36[P2505], SEQ ID NO: 37 [P2506], SEQ ID NO: 38 [P2507], SEQ ID NO: 39[P2508], or SEQ ID NO: 40 [P2509] were tested in this manner.

These feeding trials are eight days in length and consist of 96 cages,each starting with 8 male chicks. The treatments will be replicated in 8blocks, randomized within blocks of 8 cages each. A randomizationprocedure for pen assignment for treatments and blocks was used by thecontracting facility.

TABLE 8 Treatment design to test chIL10sdAB expressing corn grainAdditive Infected/ inclusion, g Cages/ Birds/ Trt DescriptionNon-Infected additive/kg feed Trt Cage T1 Nonmedicated NI 0 8 8 (NMNI)T2 Nonmedicated I 0 8 8 (NMI, NC) T3 Coccidiostat (PC) NI 0.010 8 8 T4Coccidiostat (PC) I 0.010 8 8 T5 SEQ ID NO: 135 I 4000 8 8(chIL10sdAB1A11) T6 SEQ ID NO: 135 I 4000 8 8 (chIL10sdAB1A11) T7 SEQ IDNO: 135 I 1 8 8 (chIL10sdAB1A11) T8 SEQ ID NO: 135 I 40 8 8(chIL10sdAB1A11) T9 SEQ ID NO: 135 I 500 8 8 (chIL10sdAB1A11) T10 SEQ IDNO: 135 I 300 8 8 (chIL10sdAB1A11) T11 SEQ ID NO: 135 I 150 8 8(chIL10sdAB1A11) T12 SEQ ID NO: 135 I 50 8 8 (chIL10sdAB1A11)

Other sdABs were tested using the same trial design, same grainloadings, but different chIL10sdAB expressing corn grain. In a similarway, the same trial design was used to test IL-10R peptide antagonistsas shown in Table 9.

TABLE 9 Treatment design to test IL-10R antagonist peptides Additiveinclusion, g Infected/ additive/kg Cages/ Birds/ Trt DescriptionNon-Infected feed Trt Cage T1 Nonmedicated (NMNI, NI 0 8 8 NC) T2Nonmedicated (NMI, I 0 8 8 NC) T3 Coccidiostat (PC) NI 0.010 8 8 T4Coccidiostat (PC) I 0.010 8 8 T5 P21 NI 0.070 8 8 T6 P21 I 0.070 8 8 T7P21 I 0.050 8 8 T8 P21 I 0.035 8 8 T9 P21 I 0.020 8 8 T10 P21 I 0.015 88 T11 P21 I 0.010 8 8 T12 P21 I 0.001 8 8

At the start of every trial, the facility was checked to ensure that allcages have water and feed available in each cage, which was provided toanimal ad libitum. The building temperature was maintained asappropriate for the age of the birds. Even, continuous illumination wasprovided by fluorescent lamps hung vertically along the wall. Cages willbe checked twice daily, and observations including availability of feed,water, temperature and any unusual conditions were recorded. Mortalitybirds were removed from cages, and the cage number, date, weight of thebird, sex and probable cause of death were recorded.

As part of the trial, an unmedicated commercial starter rationcompounded with basal feedstuffs was formulated. This ration was used toformulate the study's negative and positive control rations, andexperimental diets, which were all fed ad libitum from the date of chickarrival until completion of the study. Quantities of all basal feed andtest articles used to prepare treatment batches were documented andtested as part of the trial quality control procedures. Treatment dietswere mixed to a uniform distribution of test article. The mixer wasflushed between control and treatment diets, and in between eachtreatment diet. Each treatment feed was then distributed among cages ofthe corresponding treatment.

Day of hatch male chicks (Cobb 500) were used in the study. Uponarrival, chicks will be colony raised in Coccidia free battery cages. At12 days of age (trial day 0) chicks will grouped into sets of 8,weighed, and placed into an assigned cage. Birds were weighed by cage onday of trial 0 and 8.

On day of trial 2, all non-infected birds received 1 ml of distilledwater by oral pipette. All other birds will receive the coccidialinoculum diluted to a 1 ml volume and dosed by oral pipette. Theinoculum was a mixture of Eimeria acervulina, E. maxima, and E. tenellafield isolates, which produces a mild infection with all species.

Data were collected after starting the study on days 0, 2, 7, and 8. Onday 0, birds were weighed and allocated to their cages for the study. OnDay 2, designated birds were inoculated with coccidian. On Day 7,dropping pans were cleaned to prepare for droppings collection on Day 8,and subsequent analysis. On Day 8, birds were weighed by cage, alongwith the remaining feed, and fecal matter. Feces collected from eachcage were thoroughly mixed and prepared for fecal floatation, and eachsample was examined to determine the number of oocysts per gram of fecalmatter. All birds were scored for coccidian lesions on day 8 using themethod of Johnson and Reid (1970). During the trial death weights wererecorded and clinical observations were recorded twice each daythroughout the study.

Feed in-take, body weight gain, feed conversion, opgs, coccidian lesionscores, and mortality were measured for each group and analyzed bystandard statistical methods. The effect of sdAB (or peptide)supplementation was compared between groups treated with Eimeria and nottreated with Eimeria, between treatment groups treated with Eimeria andantibody (or peptide) and control groups treated and not treated withEimeria, control groups treated with Eimeria and no antibody (orpeptide) or Coccidiostat, and control groups treated with Eimeria andalso treated with a Coccidiostat.

Additionally the minimum effective dose was determined by seeing whichantibody (or peptide) dose reduced fecal oocyst counts or lesion scoresrelative to the control birds that were infected but not treated withantibody (or peptide) or Coccidiostat. Using this design the extent ofoocyst and lesion scoring reduction were determined as a function ofdose.

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The references cited throughout this application, are incorporated forall purposes apparent herein and in the references themselves as if eachreference was fully set forth. For the sake of presentation, specificones of these references are cited at particular locations herein. Acitation of a reference at a particular location indicates a manner(s)in which the teachings of the reference are incorporated. However, acitation of a reference at a particular location does not limit themanner in which all of the teachings of the cited reference areincorporated for all purposes.

It is understood, therefore, that this invention is not limited to theparticular embodiments disclosed, but is intended to cover allmodifications which are within the spirit and scope of the invention asdefined by the appended claims; the above description; and/or shown inthe attached drawings.

What is claimed is:
 1. A transgenic plant or tissues thereof comprisinga synthetic polynucleotide encoding an anti-IL-10 single domainantibody, wherein the anti-IL-10 single domain antibody comprises anamino acid sequence of SEQ ID NO: 146, and binds a polypeptidecomprising an amino acid sequence of SEQ ID NO: 80 with an EC₅₀ of 60 nMor less, as measured by ELISA.
 2. The transgenic plant or tissuesthereof of claim 1, wherein the synthetic polynucleotide comprises asequence to of SEQ ID NO: 175 or
 176. 3. The transgenic plant or tissuethereof of claim 1, wherein a plant is selected from the groupconsisting of: corn, soybean, wheat, rice, sorghum, canola, cotton, andswitchgrass.
 4. An animal feed comprising the transgenic plant or tissuethereof of claim
 1. 5. The animal feed of claim 1, wherein theanti-IL-10 single domain antibody is active upon expression in the plantand exposure to a temperature in the range from 25° C. to 130° C.
 6. Theanimal feed of claim 4 further comprising a feed supplement.
 7. Theanimal feed of claim 6, wherein the feed supplement is plant material.8. The animal feed of claim 7, wherein the plant material is anon-transgenic plant or a transgenic plant.
 9. The animal feed of claim7, wherein the plant material includes at least one component selectedfrom the group consisting of: corn meal, corn pellets, wheat meal, wheatpellets, wheat grain, barley grain, barley pellets, soybean meal,soybean oilcake, sorghum grain and sorghum pellets.
 10. The animal feedof claim 6, wherein the feed supplement includes one or more exogenousenzymes.
 11. The animal feed of claim 11, wherein the one or moreexogenous enzymes includes a hydrolytic enzyme selected from the groupconsisting of: xylanase, endoglucanase, cellulase, protease, phytase,amylase and mannanase.
 12. The animal feed of claim 6, wherein the feedsupplement includes at least one component selected from the groupconsisting of: soluble solids, fat and vermiculite, limestone, plainsalt, DL-methionine, L-lysine, L-threonine, monensin, vitamin premix,dicalcium phosphate, selenium premix, choline chloride, sodium chloride,and mineral premix.
 13. A method of treating or preventing agastrointestinal infection in an animal comprising administering to ananimal the transgenic plant or tissue thereof of claim
 1. 14. The methodof claim 13, wherein the step of administering is performed by feedingor injecting.
 15. The method of claim 13, wherein the gastrointestinalinfection is caused by a gastrointestinal pathogen selected from thegroup consisting of: a bacteria, yeast, fungi, archae, virus, andprotozoa.
 16. The method of claim 15, wherein the gastrointestinalpathogen belongs to the genus Eimeria.
 17. The method of claim 16,wherein the gastrointestinal pathogen is selected from the groupconsisting of: Eimeria tenella, Eimeria acervulina, and Eimeria maxima.18. The method of claim 13, wherein treating stimulates the immunesystem and enhances growth of an animal.
 19. The method of claim 18,wherein the animal is selected from the group consisting of: a chicken,a turkey, or a duck.